Differences in stemness properties associated with the heterogeneity of luminal-type breast cancer.

BACKGROUND: Luminal-type breast cancers are the most abundant subtype. Endocrine therapies targeting estrogen receptor (ER) or estradiol (E2) synthesis have achieved marked improvement in disease-free and overall survival of ER-positive cancers. However, approximately one-third of these cancers are poorly responsive to endocrine therapies, suggesting nonuniform tumor cell characteristics of this subtype. Recently, the tumorigenesis theory which states that CSCs are capable of self-renewal, tumorigenicity, and therapeutic resistance, became widely accepted. We investigated the relationship between the heterogeneity of luminal-type breast cancer and stemness properties. MATERIALS AND METHODS: CSC surface markers and expression of stemness-related genes, including Octamer-binding transcription factor 4 (OCT4), Nanog homeobox (NANOG), and Kruppel-like factor 4 (KLF4), were analyzed in clinical samples. ER activities were analyzed using the adenovirus vector carrying the ERE-green fluorescent protein (GFP). We separated the luminal-type breast cancers into 2 groups according to stemness-related gene expression patterns in mammospheres. RESULTS: The group that predominantly expressed NANOG mRNA showed a high percentage of the cells that were positive for CD44 and negative for CD24 and Hoechst (possessing high-stemness properties), younger patient age, higher p53 expression, and tended to show higher histological grade and higher topoisomerase IIα expression. The ERE-GFP assay revealed that the luminal-type breast cancer mammospheres were heterogeneous. Mammospheres from several specimens lacked ER activity and responsiveness to E2 but some retained ER activities. CONCLUSION: ERE activity differences might be associated with endocrine therapy effectiveness. Mammosphere stemness properties could be a useful and novel criterion for subclassification of luminal-type breast cancers.

Androgen metabolite-dependent growth of hormone receptor-positive breast cancer as a possible aromatase inhibitor-resistance mechanism.

Aromatase inhibitors (AIs) have been reported to exert their antiproliferative effects in postmenopausal women with hormone receptor-positive breast cancer not only by reducing estrogen production but also by unmasking the inhibitory effects of androgens such as testosterone (TS) and dihydrotestosterone (DHT). However, the role of androgens in AI-resistance mechanisms is not sufficiently understood. 5α-Androstane-3ß,17ß-diol (3ß-diol) generated from DHT by 3ß-hydroxysteroid dehydrogenase type 1 (HSD3B1) shows androgenic and substantial estrogenic activities, representing a potential mechanism of AI resistance. Estrogen response element (ERE)-green fluorescent protein (GFP)-transfected MCF-7 breast cancer cells (E10 cells) were cultured for 3 months under steroid-depleted, TS-supplemented conditions. Among the surviving cells, two stable variants showing androgen metabolite-dependent ER activity were selected by monitoring GFP expression. We investigated the process of adaptation to androgen-abundant conditions and the role of androgens in AI-resistance mechanisms in these variant cell lines. The variant cell lines showed increased growth and induction of estrogen-responsive genes rather than androgen-responsive genes after stimulation with androgens or 3ß-diol. Further analysis suggested that increased expression of HSD3B1 and reduced expression of androgen receptor (AR) promoted adaptation to androgen-abundant conditions, as indicated by the increased conversion of DHT into 3ß-diol by HSD3B1 and AR signal reduction. Furthermore, in parental E10 cells, ectopic expression of HSD3B1 or inhibition of AR resulted in adaptation to androgen-abundant conditions. Coculture with stromal cells to mimic local estrogen production from androgens reduced cell sensitivity to AIs compared with parental E10 cells. These results suggest that increased expression of HSD3B1 and reduced expression of AR might reduce the sensitivity to AIs as demonstrated by enhanced androgen metabolite-induced ER activation and growth mechanisms. Androgen metabolite-dependent growth of breast cancer cells may therefore play a role in AI-resistance.

Aromatase localization in human breast cancer tissues: possible interactions between intratumoral stromal and parenchymal cells.

Aromatase is a key enzyme in intratumoral estrogen production required for the production of estrogens through the conversion of serum androgens in postmenopausal breast cancer patients. There have been, however, controversies regarding the intratumoral localization of aromatase in human breast carcinoma tissues. Therefore, we have first examined the intratumoral localization of aromatase mRNA/protein in 19 breast carcinomas using laser capture microdissection/quantitative reverse transcription-PCR (RT-PCR) and immunohistochemistry. Aromatase mRNA and protein were detected in both intratumoral stromal and parenchymal cells in breast carcinoma tissues. Subsequent microarray expression profiling and clustering analyses, in addition to quantitative RT-PCR studies, showed a significant positive correlation between aromatase and estrogen-related receptor alpha mRNA expression in isolated carcinoma cells. We further examined an interaction between stromal cells isolated from human breast carcinoma tissues and breast carcinoma cell lines using a coculture system to study the biological characteristic of aromatase expression in carcinoma cells. Aromatase mRNA and enzyme activity and 17beta-hydroxysteroid dehydrogenase type 1 mRNA in breast carcinoma cell lines, including MCF-7 and SK-BR-3 cells, were up-regulated in the presence of patient-derived 32N or 74T intratumoral stromal cells. The results from steroid conversion assays were also consistent with the findings above. The results of our study also showed that aromatase inhibitors were more effective in inhibiting aromatization induced by coculture in MCF-7 than that in stromal 32N. The examination of the localization of aromatase and its regulation, including the interactions existing between different cell types in human breast carcinoma tissues, may provide important information as to achieving better clinical response to aromatase inhibitors in breast cancer patients.

Expression profiles of sex steroid receptors in desmoid tumors.

Desmoid tumors are benign fibrous neoplasms which arise from the fibrous tissue of intra- and extra- abdominal sites, but their clinical management is sometimes difficult because of extensive infiltration into the surrounding tissues. Desmoid tumors commonly occur in women, especially after childbirth. Recently, both clinical and experimental findings indicate the possible roles of sex steroids in the development and progression of desmoid tumors but detailed information is still ambiguous. In this study, we first examined immunoreactivity of sex steroid receptors in desmoid tumors (27 cases) by immunohistochemistry and compared the findings with those in reactive self-limiting lesions associated with fibrosis (8 cases). Estrogen receptor (ER) alpha and ERbeta immunoreactivities were detected in 7.4% (2/27) and 7.4% (2/27) of desmoid tumors, respectively. One desmoid tumor expressed both ERalpha and ERbeta. Progesterone receptor (PR)-A and PR-B were detected in 25.9% (7/27) and 33.3% (9/27), respectively, and androgen receptor (AR) in 52.9% (14/27). In reactive lesions with fibrosis, only AR was detected in 37.5% (3/8). Sex steroid receptor mRNAs was further examined by reverse transcription and polymerase chain reaction (RT-PCR) analysis using fresh frozen tissues, demonstrating the expression of PR (PR-A and/or PR-B) and AR mRNAs in eight desmoid tumors examined and all cases of reactive fibrosis. These results indicate that sex steroid hormones might play an important role in the pathogenesis of desmoid tumors and could lead to the introduction of novel hormone therapeutic approaches in managing patients with recurrent desmoid tumors.

Progesterone receptor subtypes in vascular smooth muscle cells of human aorta.

Progesterone is involved in various functions of the cardiovascular system, including those of vascular smooth muscle cells (VSMCs) via progesterone receptor (PR). Progesterone has also been postulated to be involved in inhibition of VSMC proliferation via PR. However, the details of PR expression have remained largely unknown in human cardiovascular VSMCs. Therefore, we first examined the relative levels of PR isoform (PR-A and PR-B) expression in VSMCs, using both immunohistochemistry and quantitative RT-PCR analysis. PR-B was equally expressed between male and female aorta, but PR-A was more abundant in female than in male aorta. This finding demonstrated that the status of PR subtype expression was associated with the difference of genders.

Analysis of gene expression induced by diethylstilbestrol (DES) in human primitive Mullerian duct cells using microarray.

The Mullerian ducts are strongly influenced by natural estrogen, estradiol (E2) and diethylstilbestrol (DES) in their development. We screened E2 and DES responsive genes using a microarray analysis in human primitive Mullerian duct cell line, EMTOKA cells expressed estrogen receptor (ER) beta. c-myc oncogene and other target genes expression was detected in cells treated by high-dose DES, but ER antagonist ICI 182,780 could not prevent c-myc induction above. Results of our present study suggested the presence of ER independent pathway in oncogenes induction process by high-dose DES treatment in a human primitive Mullerian duct cell line.

3beta-Hydroxysteroid dehydrogenase in human aorta.

3beta-Hydroxysteroid dehydrogenase (3beta-HSD) is known to be involved in steroid production and/or metabolism and to be expressed in many tissues including adrenal cortex. Expression of this enzyme has also been elucidated in human cardiovascular system but its details remain largely unknown. Therefore, in this study, we examined the status of 3beta-HSD in postmortem human aorta utilizing RT-PCR and immunohistochemical analysis. Both mRNAs and immunoreactivity for the enzyme were detected predominantly in female aorta, and in those with mild atherosclerotic changes. In addition, immunohistochemical study demonstrated that immunoreactivity for 3beta-HSD was detected in vascular smooth muscle cells (VSMCs) of aorta. These findings all indicated that steroidogenesis via 3beta-HSD may occur in VSMCs of human aorta, possibly related to gender differences and/or the degree of atherosclerosis.

Steroid and xenobiotic receptor (SXR), cytochrome P450 3A4 and multidrug resistance gene 1 in human adult and fetal tissues.

The steroid and xenobiotic receptor (SXR) has been demonstrated to play an important role in the regulation of the cytochrome P450 3A4 gene (CYP3A4) and multidrug resistance gene 1 (MDR1) by both endogenous and xenobiotic substrates. SXR and its rodent ortholog PXR exhibit marked differences in their ability to be activated by xenobiotic inducers. This suggests that results obtained by rodent models may not always accurately predict responses to the same compounds in humans. SXR expression was demonstrated in the human liver and intestine, but its systemic distribution remains unknown. Therefore in this study, we first characterized the expression of SXR and its target genes CYP3A4, and MDR1 in human adult and fetal tissues using quantitative RT-PCR, immunoblotting, and combined laser capture microscopy and RT-PCR analysis. SXR mRNA and protein are expressed in adult and fetal liver, lung, kidney, and intestine. There is a close association between the expression of SXR and its target genes in all of the tissues examined. The amounts of SXR mRNA in the liver and intestine reached maximal levels in young adults (15-38 years old) and then subsequently decreased to less than half of the maximal levels with aging. These findings demonstrated age-related differences in the body's capacity to metabolize steroids and xenobiotic compounds and suggest an important role for SXR and its target genes, CYP3A4 and MDR1 in this process.

E4F1, a novel estrogen-responsive gene in possible atheroprotection, revealed by microarray analysis.

Estrogen has been postulated to be involved in inhibition of vascular smooth muscle cell (VSMC) proliferation mainly via estrogen receptor (ER), but the detailed mechanism has remained primarily unknown. Therefore, in this study, microarray analysis was used in two types of cultured human VSMCs: one positive for ER alpha, and the other for ER beta, which were treated by estrogens to detect the estrogen-responsive genes. We also used quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to evaluate mRNA levels of selective target gene (TG) in these cells. We further studied whether the TG product was involved in inhibition of proliferation using small interfering RNA (siRNA) of the TG transfection. We subsequently used quantitative RT-PCR and in situ hybridization analysis to evaluate the expression of these gene products in human aorta. E4F1, a possible inducer of cell growth arrest, was markedly increased only in ER alpha-positive VSMCs by estrogens in both microarray and RT-PCR analyses. Blocking of E4F1 using siRNA suppressed estrogenic inhibition of ER alpha-positive VSMC proliferation. E4F1 mRNA was abundant in premenopausal female aorta with mild atherosclerotic changes. E4F1 is therefore considered one of the estrogen-responsive genes involving ER alpha-mediated inhibition of VSMC proliferation and may play an important role in estrogen-related atheroprotection of human aorta.

Estrogen receptors in atherosclerotic human aorta: inhibition of human vascular smooth muscle cell proliferation by estrogens.

Estrogen has been postulated to exert direct anti-atherogenic effects via binding to estrogen receptors (ERs) in vascular smooth muscle cells (VSMCs). Therefore, we believe it is important to examine the status of ER expression in the human cardiovascular system and its disorders. In this study, we first evaluated the relative abundance of messenger RNA (mRNA) of both ER subtypes (ERalpha and ERbeta) in the human aorta using reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR). We then examined the immunolocalization of both ERs in VSMCs of human atherosclerotic lesions. In order to examine which ER subtype was associated with the anti-atherogenic effects of estrogen, we examined the effects of estrogen in two VSMC cell lines, one positive only for ERalpha and the other positive only for ERbeta. The relative abundance of mRNAs for both ERs was higher in female aorta with a mild degree of atherosclerosis than in female aorta with a severe degree of atherosclerosis (P < 0.05). In addition, the number of ERalpha and/or ERbeta double positive cells in the neointima was higher in female aorta with a mild degree of atherosclerosis than in female aorta with severe atherosclerosis (P < 0.05). Our in vitro study found that estradiol was able to significantly inhibit the proliferation of ERalpha positive VSMCs but not ERbeta positive VSMCs (P < 0.05). Moreover, estradiol was found to significantly suppress proliferating cell nuclear antigen (PCNA) mRNA levels in ERalpha positive VSMCs compared to that of ERbeta positive VSMCs, consistent with the findings of cell proliferation. Results from this study suggest that estrogens can inhibit the proliferation of VSMCs through ERalpha, especially in pre-menopausal women. Our study also indicates that decreased levels of ER, especially ERalpha, in the female atherosclerotic neointima may be associated with progression of atherosclerotic changes.

Sex steroid receptors in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a disease characterized primarily by chronic inflammatory synovitis and is well-known to be associated with significant sex differences in its prevalence and clinical features. Sex steroids have been proposed to be involved in the pathogenesis of RA, but details pertaining to the expression of sex steroid receptors in RA synovial tissue have yet to be fully characterized. In the present study, we examined oestrogen receptor (ER) alpha, ERbeta, progesterone receptor (PR) and androgen receptor (AR) mRNA expression using real-time reverse transcriptase-PCR (RT-PCR) in eight female RA synovial tissues and six female synovial tissues without inflammation, and determined immunolocalization of ERalpha, ERbeta, PR-A, PR-B and AR using immunohistochemistry in synovial tissues obtained from 22 RA patients. Real-time RT-PCR analysis demonstrated the expression of ER, PR and AR mRNAs in both RA and non-inflamed synovial tissues. Relative abundance of ER mRNAs was significantly higher in RA synovial tissue than non-inflamed synovial tissue (P<0.05). In addition, the relative ERalpha/ERbeta mRNA expression ratio was significantly lower in RA than non-inflamed synovial tissue (RA, 2.34 +/- 1.60; and non-inflamed, 20.7 +/- 19.1; P<0.05). There were no significant differences in relative abundance of PR mRNA. Relative abundance of AR mRNA was significantly lower in RA (P<0.05). Immunoreactivity for ERalpha, ERbeta, PR-B and AR was detected in the lining cells, inflammatory cells and fibroblasts in all the patients examined. The labelling indices for ERbeta and PR-B were more abundant in both lining cells (ERbeta, 54.2 +/- 12.2%; PR-B, 73.6 +/- 18.9%) and inflammatory cells (ERbeta, 74.6 +/- 16.2%; PR-B, 75.9 +/- 16.1%) than in fibroblasts (ERbeta, 36.5 +/- 15.6%; PR-B, 49.4 +/- 18.0%). Labelling indices for ERalpha and AR were significantly higher in lining cells (ERalpha, 14.4 +/- 8.6%; AR, 31.2 +/- 11.3%) and fibroblasts (ERalpha, 12.1 +/- 7.5%; AR, 20.1 +/- 9.6%) than those in inflammatory cells (ERalpha, 5.7 +/- 3.3%; AR, 9.2 +/- 4.4%). There were significant differences (P<0.05) in the labelling indices for ERalpha, ERbeta and PR-B between men and women under 50 years of age in fibroblasts of RA synovial tissues. These results indicate that sex steroid receptors are present in RA and non-inflamed synovial tissues, including inflammatory cells in RA, and suggest that sex steroids may play important roles in the regulation of inflammation of RA synovial tissue.

Steroid sulfatase and estrogen sulfotransferase in the atherosclerotic human aorta.

Various epidemiological studies have demonstrated a relatively low incidence of cardiovascular events in premenopausal women and its marked increment after menopause. In addition, estrogens have been postulated to exert direct anti-atherogenic effects via binding to estrogen receptors in vascular smooth muscle cells (VSMCs). However, not all postmenopausal women develop atherosclerosis despite decreased levels of serum estrogen. Therefore, we believe it is important to examine the status of estrogen metabolism in situ in the human cardiovascular system. Estrone sulfate (E1S) is a major circulating plasma estrogen that is converted into the biologically active estrogen, estrone (E1) by steroid sulfatase (STS). E1 is also sulfated and reverted into E1S by estrogen sulfotransferase (EST). These two enzymes have recently been shown to play important roles in the in situ estrogen actions of estrogen-dependent human tissues and various sex steroid-dependent tumors. STS and EST, however, have not been studied in detail in the human vascular system associated with atherosclerotic changes. In the present study, we evaluated the relative abundance of STS- and EST-immunoreactive protein and mRNA expression in human aorta using immunohistochemistry and reverse transcription followed by quantitative polymerase chain reaction in addition to enzyme activity. STS expression levels were found to be significantly higher in the VSMCs obtained from female aortas with mild atherosclerotic changes than in those with severe atherosclerotic changes and in male aortas regardless of atherosclerotic changes. EST expression levels in the VSMCs of these aortas, however, were significantly higher in female aortas with severe atherosclerotic changes and in male aortas than in female aortas with mild atherosclerotic changes. We believe it is important to examine factors regulating the expression and activity of these estrogen-metabolizing enzymes in the human aorta. Various cytokines have been proposed to function as regulators of these enzymes in other tissues. In the present study, we studied the effects of interleukin (IL)-1beta, known to be produced in human atherosclerotic lesions, on the expression of these enzymes using cultured human VSMCs originally obtained from a female patient. IL-1beta markedly inhibited the expression of STS mRNA and enzyme activity, but stimulated the expression of EST mRNA and enzyme activity. In addition, IL-1beta also reduced E2 production from E1S and E1 in VSMCs. Results from the present study seem to suggest that the expression levels of both STS and EST mRNA and activity may be significantly associated with the degree of atherosclerotic changes in the female aorta, which may be related to cytokines produced in situ, such as IL-1beta, in human atherosclerotic lesions.

The possible roles of mineralocorticoid receptor and 11beta-hydroxysteroid dehydrogenase type 2 in cardiac fibrosis in the spontaneously hypertensive rat.

In hypertension, aldosterone has been demonstrated to play a crucial role in cardiac fibrosis, which generally increases cardiac morbidity and death. However, few studies have reported the expression of the mineralocorticoid receptor (MR) and 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) in the heart under hypertensive conditions. Therefore, in this study, spontaneously hypertensive rats (SHR) were examined to elucidate the possible actions of mineralocorticoids via binding to MR. Wister Kyoto Rat (WKY), SHR, stroke-prone SHR (SHRSP), and malignant SHRSP (M-SHRSP) were used. Total RNA was extracted from the left ventricle of these rats, and examined for the expression levels of MR, 11beta-HSD2 and Collagen types 1 and 3 using reverse transcription real-time quantitative polymerase chain reaction employing the Light Cycler Instrument. Blood pressure was significantly different among each group. The mean mRNA levels for MR, 11beta-HSD2 and Collagen types 1 and 3 in M-SHRSP were found to be significantly increased compared to those of WKY, whereas no significant differences in mRNA levels were detected among SHR and SHRSP. Findings from the present study appear to demonstrate that MR and 11beta-HSD2 mRNA significantly rise in the left ventricle of M-SHRSP and increase of these mRNA is one of the cause of cardiac fibrosis.