Shuji Nagasaki

Intratumoral Estrogens and Estrogen Receptors in Human Non–Small Cell Lung Carcinoma

Purpose: The possible involvement of gender-dependent factors has been suggested in human non-small cell lung carcinomas (NSCLC), but their precise roles remain largely unclear. Therefore, we examined intratumoral estradiol concentrations in NSCLC to examine local actions of estrogens in NSCLC. Experimental Design: Fifty-nine frozen specimens of NSCLC were available for liquid chromatography/electrospray tandem mass spectrometry to study intratumoral estradiol concentrations. In addition, A549 NSCLC cells stably expressing estrogen receptor (ER) α (A549 + ERα) or ERβ (A549 + ERβ) were used in vitro studies. Results: Forty-three (73%) of 59 NSCLC showed higher concentration of estradiol in carcinoma tissues than the corresponding nonneoplastic lung tissues from the same patient, and intratumoral estradiol concentrations were significantly (P = 0.0002 and 2.2-fold) higher than the corresponding nonneoplastic lungs. The intratumoral concentration of estradiol was positively correlated with aromatase expression, tumor size, and Ki-67 status in ERα- or ERβ-positive cases. In in vitro studies, estradiol significantly increased cell proliferation of A549 + ERα or A549 + ERβ, which was significantly suppressed by selective ER modulators, tamoxifen or raloxifene. Both A549 + ERα and A549 + ERβ cells expressed aromatase. The cell proliferation level in these cells was significantly increased under treatment with testosterone, and it was inhibited by addition of the aromatase inhibitor letrozole. Conclusions: These results suggest that estradiol is locally produced in NSCLC mainly by aromatase and plays an important role in the growth of ERα- or ERβ-positive NSCLC. Therefore, use of selective ER modulators and/or aromatase inhibitors may be clinically effective in NSCLC that are positive for both ER and aromatase.

In situ estrogen production and its regulation in human breast carcinoma : From endocrinology to intracrinology

The great majority of breast carcinomas arising in postmenopausal women are estrogen dependent or positive for estrogen receptor (ER) in carcinoma cells despite markedly low plasma or circulating estrogen concentrations. In these patients, biologically active estrogens are locally produced from circulating inactive steroids including adrenal androgens in an intracrine mechanism in the breast cancer tissues and confer estrogenic activities on carcinoma cells. A series of enzymes are involved in this intra‐tumoral or in situ production of estrogens in breast carcinoma tissues but aromatase, a member of the cytochrome P450 family, is a key enzyme of estrogen production through conversion from circulating adrenal androgens in estrogen‐dependent postmenopausal breast cancer. It then becomes important to identify the sites of this estrogen production. There has been, however, controversy regarding intra‐tumoral localization of aromatase in breast carcinoma, especially whether intra‐tumoral production of estrogens through aromatase occurs in carcinoma or stromal cells. The enzyme was demonstrated to be expressed in both carcinoma and stromal cells in breast carcinoma tissues on immunohistochemistry with a well‐characterized mAb 677 and combined laser capture microdissection/qualitative reverse transcriptase–polymerase chain reaction. Intra‐tumoral aromatase in both of these cell types was subsequently demonstrated to be induced by carcinoma–stromal interactions associated with carcinoma invasion in breast tissue. The signals through various nuclear receptors, especially estrogen‐related receptor‐α in carcinoma cells and liver receptor homologue‐1 in adipocytes adjacent to carcinoma invasion, in conjunction with various cytokines and/or growth factors, play pivotal roles in this induction of intra‐tumoral aromatase. This increased aromatase subsequently results in increased in situ estrogen concentrations of breast cancer. Aromatase inhibitors are currently established as the gold standard for the treatment for ER‐positive breast carcinoma but resistance to the therapy still remains to be solved by other modes of suppression of intra‐tumoral estrogen production.

Increased intratumoral androgens in human breast carcinoma following aromatase inhibitor exemestane treatment

Sex steroids play important roles in the development of many human breast carcinomas, and aromatase inhibitors are used for the anti-estrogen therapy. Recent studies have demonstrated that aromatase suppressed 5alpha-dihydrotestosterone (DHT) synthesis in breast carcinoma cells, but intratumoral concentration of androgens and its significance have not been reported in the breast carcinoma patients treated with aromatase inhibitors. Therefore, we examined androgen concentrations in breast carcinoma tissues treated with exemestane, and further performed in vitro studies to characterize the significance of androgen actions. Intratumoral DHT concentration was significantly higher in breast carcinoma tissues following exemestane treatment (n=9) than those without the therapy (n=7), and 17beta-hydroxysteroid dehydrogenase type 2 (17betaHSD2) status was significantly altered to be positive after the treatment. Following in vitro studies showed that 17betaHSD2 expression was dose dependently induced by both DHT and exemestane in T-47D breast carcinoma cells, but these inductions were not additive. DHT-mediated induction of 17betaHSD2 expression was markedly suppressed by estradiol (E(2)) in T-47D cells. E(2)-mediated cell proliferation was significantly inhibited by DHT in T-47D cells, associated with an increment of 17betaHSD2 expression level. These findings suggest that intratumoral androgen actions are increased during exemestane treatment. 17betaHSD2 is a potent DHT-induced gene in human breast carcinoma, and may not only be involved in anti-proliferative effects of DHT on breast carcinoma cells but also serve as a potential marker for response to aromatase inhibitor in the breast carcinoma patients.

17β-Hydroxysteroid Dehydrogenase Type 12 in Human Breast Carcinoma: A Prognostic Factor via Potential Regulation of Fatty Acid Synthesis

17beta-Hydroxysteroid dehydrogenase type 12 (17beta-HSD12) has been shown to be involved in elongation of very long chain fatty acid (VLCFA) as well as in biosynthesis of estradiol (E2). 17beta-HSD12 expression was also reported in breast carcinomas but its functions have remained unknown. In this study, we examined the correlation between mRNA expression profiles determined by microarray analysis and tissue E2 concentrations obtained from 16 postmenopausal breast carcinoma cases. No significant correlations were detected between 17beta-HSD12 expression and E2 concentration. We then immunolocalized this enzyme in 110 cases of invasive ductal carcinoma. 17beta-HSD12 immunoreactivity in breast carcinoma cells was significantly associated with poor prognosis of the patients. We further examined the biological significance of 17beta-HSD12 using cell-based studies. Small interfering RNA-mediated knockdown of 17beta-HSD12 in SK-BR-3 (estrogen receptor-negative breast carcinoma cell line) resulted in significant growth inhibition, which was recovered by the addition of VLCFAs such as arachidonic acid. The status of 17beta-HSD12 immunoreactivity was also correlated with adverse clinical outcome in cyclooxygenase 2 (COX2)-positive breast cancer patients but not in COX2-negative patients. Therefore, these findings indicated that 17beta-HSD12 was not necessarily related to intratumoral E2 biosynthesis, at least in human breast carcinoma, but was rather correlated with production of VLCFAs such as arachidonic acid, which may subsequently be metabolized to prostaglandins by COX2 and result in tumor progression of the patients.

17β-Hydroxysteroid Dehydrogenases in Human Breast Cancer

Estrogen plays a pivotal role in development and progression of human breast carcinoma. Before menopause the main source of estrogen in women is circulating estrogen secreted from the ovary, but following menopause the source changes to the hormone that is converted from circulating adrenal androgens in peripheral tissues. Therefore, adrenal androgens have to be converted to estrogen to stimulate breast carcinoma cells. In these steps, several enzymes such as aromatase, steroid sulfatase, and 17β‐hydroxysteroid dehydrogenases (17β‐HSDs) are involved in the production of estrogens.

Sex steroid receptors expression and hormone‐induced cell proliferation in human osteosarcoma

Sex steroid receptors including estrogen receptors (ER), progesterone receptors (PR), and androgen receptors (AR) have been sporadically reported in human osteosarcoma or its cell lines. Therefore, sex steroids have been considered to play some roles in human osteosarcoma, but no systematic and detailed studies regarding the correlation between the status of these receptors in sarcoma cells and clinicopathological parameters have been reported. We examined the existence of ER, PR and AR in 28 cases of osteosarcoma using immunohistochemistry. We then characterized the potential influence of sex steroids on cell proliferation of osteosarcoma cells using MG‐63 human osteosarcoma cell line, which expressed all of these receptors. ER‐β and PR were detected in the great majority of the cases (23 and 24 cases, respectively) but ER‐α and aromatase were not detected in all the cases, and AR was detected only in eight cases. There was a significant positive correlation between ER‐β and Ki‐67 (MIB1) labeling indexes. The absence of aromatase in tumors also suggests the relative importance of concentrations of circulating sex steroids. Proliferation of MG‐63 cells was significantly stimulated by estradiol, progesterone, and 5α‐dihydrotestosterone (DHT), and was significantly suppressed by the addition of fulvestrant (ICI), mifepristone (RU), and hydroxiflutamide, blockers for ER, PR and AR, respectively. Sex steroids, particularly estrogen and progesterone, are considered to play important roles in the regulation of cell proliferation in human osteosarcoma. In addition, these data suggest the potential for a novel endocrine therapy in osteosarcoma using clinically available inhibitors of progesterone and estrogen actions. (Cancer Sci 2008; 99: 518–523)

Chicken ovalbumin upstream promoter transcription factor II in human breast carcinoma: Possible regulator of lymphangiogenesis via vascular endothelial growth factor‐C expression

Chicken ovalbumin upstream promoter transcription factors (COUP‐TF) are orphan members of the nuclear receptor superfamily and consist of COUP‐TFI and COUP‐TFII. COUP‐TFI was reported to be overexpressed in human breast cancer and to promote estrogen‐independent transcriptional activity of estrogen receptor α. COUP‐TFII, however, has not been examined in the breast. Therefore, we carried out immunohistochemical analysis of COUP‐TFII in human breast cancer in order to clarify its biological and clinical significance. We immunolocalized COUP‐TFII in 119 human breast cancers and correlated the findings with various clinicopathological parameters. Fifty‐nine percent of the cases were immunohistochemically positive for COUP‐TFII. COUP‐TFII positivity was correlated with poor clinical outcome, and a statistically significant correlation was detected between COUP‐TFII and the following clinicopathological parameters: clinical stage, lymph node status, histological grade, and estrogen receptor α status. In addition, short interfering RNA‐mediated knockdown of COUP‐TFII in the breast carcinoma cell line MCF‐7 decreased the level of vascular endothelial growth factor‐C mRNA expression, which is a known inducer of lymphangiogenesis and lymph node metastasis. These results suggest that COUP‐TFII is involved in the development of advanced human breast cancer. (Cancer Sci 2009; 100: 639–645)

New developments in intracrinology of human breast cancer: estrogen sulfatase and sulfotransferase.

Steroid sulfatase (STS) hydrolyses biologically inactive estrogen sulfates to active estrogens, while estrogen sulfotransferase (EST) sulfonates estrogens to estrogen sulfates. Information regarding the expression of STS in human breast carcinoma tissues is still very limited compared to that of aromatase or 17β‐hydroxysteroid dehydrogenases (17β‐HSDs). In our study, EST and STS immunoreactivity was detected in carcinoma cells in 50 and 84 out of 113 breast carcinomas (44.2% and 74.3%, respectively), which was also associated with mRNA levels determined by RT/real‐time PCR. Using microdissection/RT‐PCR analyses, EST mRNA was localized to both carcinoma and intratumoral stromal cells, whereas STS was detected only in carcinoma or parenchymal cells. STS immunoreactivity was positively associated with tumor size. EST immunoreactivity was inversely correlated with tumor size or lymph node status and was significantly associated with a decreased risk of recurrence and improved prognosis. These data suggest that both EST and STS play important roles in the regulation of in situ estrogen production in human breast cancer. In addition, EST is an independent prognostic factor in human breast carcinoma and loss of EST may result in altered estrogen metabolism in hormone‐dependent breast cancer cells. An inhibition of intratumoral STS in the patients with estrogen‐dependent breast carcinoma is also considered to provide more clinical benefits, especially to the patients in which primary source of an availability of intratumoral estrogen is through STS rather than aromatase.

Co-expression of estrogen receptor beta and aromatase in Japanese lung cancer patients: gender-dependent clinical outcome.

AIM The potential gender differences in lung cancer development have been proposed on the basis of hormonal actions. We aimed to evaluate whether estrogen receptors (ERs) in non-small cell carcinoma (NSCLC) patients may primarily depend upon intratumoral estrogen produced via aromatase pathway. MAIN METHODS We evaluated ER beta (ERβ) and aromatase status in 169 Japanese NSCLC patients through immunohistochemistry analysis (IHC). Significance of IHC was further confirmed in NSCLC cell lines via in vitro assays. KEY FINDINGS IHC analysis of NSCLC patients demonstrated that both ERβ and aromatase were highly co-expressed (p=0.032) in carcinoma cells. Overall survival in males was significantly worse than that in postmenopausal female among double positive NSCLC patients (p=0.010) but not in non-double positive patients. In addition, among double positive cases, overall survival of males was significantly worse than that of postmenopausal females in those with higher ERβ Allred score ≥5, (p=0.034), but not in those with lower ERβ Allred score=3-4. In-vitro analysis demonstrated aromatase activity on testosterone treatment, which resulted in in situ estrogen production (p<0.0001) and increased proliferation of ERβ overexpressing A549 cells (p<0.0001). Aromatase inhibitor i.e. letrozole abrogated this proliferation and also enhanced the androgenic activity (p<0.0001). Testosterone treatment resulted in estrogen responsive elements activation (p<0.0001) in ERβ vector transfected A549 and LK87 cells whereas ER blocker i.e. fulvestrant abrogated this effect, (p<0.0001). SIGNIFICANCE Our results suggest that co-expression of ERβ and aromatase in NSCLCs of Japanese males may result in tumor progression and potential endocrine therapy may confer therapeutic benefits to these patients.

Comparative effects of raloxifene, tamoxifen and estradiol on human osteoblasts in vitro: Estrogen receptor dependent or independent pathways of raloxifene

SERMs bind to both estrogen receptor (ER)alpha and beta, resulting in tissue dependent estrogen agonist or antagonist responses. Both raloxifene and tamoxifen are most frequently used SERMs and exert estrogen agonistic effects on human bone tissues, but the details of their possible direct effects on human bone cells have remained largely unknown. In our present study, we examined the comparative effects of raloxifene, tamoxifen, and native estrogen, estradiol on human osteoblast cell line, hFOB in vitro. Both the cell numbers and the ratio of the cells in S phase fraction were significantly increased by the treatment of raloxifene or tamoxifen as well as estradiol treatments in hFOB. Gene profile patterns following treatment with raloxifene, tamoxifen, and estradiol demonstrated similar patterns in a microarray/hierarchal clustering analysis. We also examined the expression levels of these genes detected by this analysis using quantitative RT-PCR. MAF gene was induced by raloxifene treatment alone. GAS6 gene was induced by raloxifene and tamoxifen as well as estradiol. An estrogen receptor blocker, ICI 18, 286, inhibited an increase of GAS6 gene expression but not the levels of MAF gene mRNA expression. Results of our present study demonstrated that raloxifene exerted direct protective effects on human osteoblasts in both estrogen receptor dependent and independent manners.