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Supplementary Figure 1 from Steroid Sulfatase and Estrogen Sulfotransferase in Colon Carcinoma: Regulators of Intratumoral Estrogen Concentrations and Potent Prognostic Factors

Supplementary Figure 1 from 17β-Hydroxysteroid Dehydrogenase Type 12 in Human Breast Carcinoma: A Prognostic Factor via Potential Regulation of Fatty Acid Synthesis

Supplementary Figure 2 from 17β-Hydroxysteroid Dehydrogenase Type 12 in Human Breast Carcinoma: A Prognostic Factor via Potential Regulation of Fatty Acid Synthesis

Supplementary Figure Legends 1-2 from 17β-Hydroxysteroid Dehydrogenase Type 12 in Human Breast Carcinoma: A Prognostic Factor via Potential Regulation of Fatty Acid Synthesis

Supplementary Figure 2 from Steroid Sulfatase and Estrogen Sulfotransferase in Colon Carcinoma: Regulators of Intratumoral Estrogen Concentrations and Potent Prognostic Factors

17β-Hydroxysteroid dehydrogenase type 12 (17β-HSD12) has been shown to be involved in elongation of very long chain fatty acid (VLCFA) as well as in biosynthesis of estradiol (E2). 17β-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 17β-HSD12 expression and E2 concentration. We then immunolocalized this enzyme in 110 cases of invasive ductal carcinoma. 17β-HSD12 immunoreactivity in breast carcinoma cells was significantly associated with poor prognosis of the patients. We further examined the biological significance of 17β-HSD12 using cell-based studies. Small interfering RNA–mediated knockdown of 17β-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 17β-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 17β-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. [Cancer Res 2009;69(4):1392–9]

Previous epidemiologic and in vitro studies have indicated a potential involvement of estrogens in the pathogenesis of human colon carcinoma, but the precise roles of estrogens have remained largely unknown. Therefore, in this study, we first measured intratumoral concentrations of estrogens in 53 colon carcinomas using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS). Tissue concentrations of total estrogen [estrone (E1) + estradiol] and E1 were significantly (2.0- and 2.4-fold, respectively) higher in colon carcinoma tissues than in nonneoplastic colonic mucosa (n = 31), and higher intratumoral concentrations of total estrogen and E1 were significantly associated with adverse clinical outcome. Intratumoral concentration of total estrogen was significantly associated with the combined status of steroid sulfatase (STS) and estrogen sulfotransferase (EST), but not with that of aromatase. Thus, we subsequently examined the STS/EST status in 328 colon carcinomas using immunohistochemistry. Immunoreactivities for STS and EST were detected in 61% and 44% of the cases, respectively. The −/+ group of the STS/EST status was inversely associated with Dukes' stage, depth of invasion, lymph node metastasis, and distant metastasis and positively correlated with Ki-67 labeling index of the carcinomas. In addition, this −/+ group had significantly longer survival, and a multivariate analysis revealed the STS/EST status as an independent prognostic factor. Results from our present study showed that the STS/EST status of carcinoma tissue determined intratumoral estrogen levels and could be a significant prognostic factor in colon carcinoma, suggesting that estrogens are locally produced mainly through the sulfatase pathway and play important roles in the progression of the disease. [Cancer Res 2009;69(3):914–22]

Supplementary Table 1 from 17β-Hydroxysteroid Dehydrogenase Type 12 in Human Breast Carcinoma: A Prognostic Factor via Potential Regulation of Fatty Acid Synthesis

Supplementary Table 1 from Steroid Sulfatase and Estrogen Sulfotransferase in Colon Carcinoma: Regulators of Intratumoral Estrogen Concentrations and Potent Prognostic Factors

We have developed a tailless, two-winged flapping drone with a full span length of 180 mm and a total weight of 20.5 g. The developed flapping drone is characterized by three biomimetic techniques: an anisotropic vein pattern reinforcing the wing surfaces, an elastic flapping mechanism, and gravity center position control in the abdomen. On the basis of experimental and numerical results, the flapping wings are reinforced by a vein pattern made of an anisotropic carbon fiber-reinforced plastic (CFRP) laminate to passively provide appropriate aeroelastic deformation and positively utilize snap-though buckling on the wing surface at stroke reversals to provide a fast feathering rotation. The flapping wing kinematics are provided by a novel flapping mechanism with an energy recovery system using the elasticity of the mechanical system. Unlike other previously developed flapping robots, feedback control to stabilize the pitch and roll angles of the drone’s body is conducted using a technique of gravity center position control, where the tail angles of the body are changed similarly to the abdominal movements of insects in flight. The developed flapping drone has succeeded in an autonomous hovering flight for more than 30 s and a vertical take-off under a wireless condition with the gravity center position control., Sensors and Materials, 33(3), pp. 859-872; 2021

The authors have developed a system for the Antarctic stratospheric aerosol observation and sample-return using the combination of a rubber balloon, a parachute, and a gliding fixed-wing unmanned aerial vehicle (UAV). A rubber balloon can usually reach 20 km to 30 km in altitude, but it becomes difficult for the UAV designed as a low-subsonic UAV to directly glide back from the stratospheric altitudes because the quantitative aerodynamic characteristics necessary for the control system design at such altitudes are difficult to obtain. In order to make the observation and sample-return possible at such higher altitudes while avoiding the problem with the control system of the UAV, the method using the two-stage separation was developed and attempted in Antarctica. In two-stage separation method, the UAV first descends by a parachute after separating from the balloon at stratospheric altitude to a certain altitude wherein the flight control system of the UAV works properly. Then it secondly separates the parachute for autonomous gliding back to the released point on the ground. The UAV in which an optical particle counter and an airborne aerosol sampler were installed was launched on January 24, 2015 from S17 (69.028S, 40.093E, 607 m MSL) near Syowa Station in Antarctica. The system reached 23 km in altitude and the UAV successfully returned aerosol samples. In this paper, the details of the UAV system using the two-stage separation method including the observation flight results, and the preliminary results of the observation and analyses of the samples are shown.

The 11th Symposium on Polar Science/Interdisciplinary sessions: [IW] Whole atmosphere, Poster presentations, Mon. 16 Nov. - Fri. 18 Dec.

TSH is the important regulator of thyroid function but detailed molecular mechanisms have not been clarified. We first generated the iodine deficient (ID) rat in which goiter is induced by accelerated endogenous TSH secretion. The result of microarray analysis demonstrated markedly increased levels of adrenomedullin 2/intermedin (AM2/IMD) expression in the ID rat thyroid. AM2/IMD is a potent vasodilator. AM2/IMD mRNA expression was induced by TSH in a rat thyroid follicular cell line FRTL-5. Immunohistochemical analysis in human normal and Graves' disease thyroid revealed that AM2/IMD immunoreactivity was detected in follicular cells and more pronounced in Graves' disease. These results indicated that TSH induced AM2/IMD expression in the rat thyroid gland and it could locally work as a potent vasodilator, resulting in the expansion of thyroid inter-follicular capillaries. AM2/IMD could also contribute to facilitate thyroid hormone synthesis possibly via vasodilation effects and/or cAMP stimulating effects in the human thyroid gland.

The antithyroid drugs (ATDs) methimazole (MMI) and propylthiouracil (PTU) have been used for treatment of hyperthyroidism for more than several decades, despite the fact that they are associated with adverse drug reactions that are thought to be autoimmune mediated. We therefore examined histopathologic responses in the immune system in male and female rats given MMI (2, 20 and 200 mg/kg/day, p.o., in experiment 1; 200 mg/kg/day, p.o., in experiment 3) or PTU (25 and 250 mg/kg/day, p.o., in experiment 2; 200 mg/kg/day, p.o., in experiment 3) for two weeks. In experiments 1 and 2, highest doses of MMI and PTU induced histopathologic changes in the spleen consistent with those in experiment 3 without any changes in the other peripheral lymphoid organs and tissues. In experiment 3, histopathological evaluation of the spleen along with hematological and bone marrow examinations were performed. In both male and female rats, MMI or PTU induced histopathological changes in the spleen characterized by development of germinal centers and an increase in the number of IgG-positive plasma cells in the red pulp; these changes were most prevalent in the MMI-treated female rats. Total red and white blood cell counts were decreased in the MMI-treated male and female rats; lymphocytes and monocytes were lower in male and female rats, respectively. Bone marrow nucleated cells were significantly lower in the MMI-treated males. This is the first study to demonstrate that ATDs induce spleen specific B-cell reactions in rats.

Bisphenol A, one of the industrial chemicals used in plastics and in the coating of dishes and medical equipment, behaves as an endocrine disruptor in the human body. Bisphenol A can bind directly to several types of nuclear receptors, including steroid and xenobiotic receptor (SXR). SXR plays an important role in bone metabolism through the activation of osteoblasts in vitro, but SXR protein localization has not been reported in bone tissues. Additionally, it is not known whether bisphenol A acts on osteoblasts through SXR activation. Therefore, in this study, we first examined the immunolocalization of the SXR protein in human adult and fetal bone tissues. We then examined the effects of bisphenol A on human osteoblasts in vitro. SXR immunoreactivity was detected in osteoblasts, but not in osteoclasts, of both adult and fetal bone tissues. In fetal bone tissues, the mesenchymal cells or fetal connective tissue were also positive for SXR immunoreactivity. Expression of SXR target genes (tsukushi, matrilin-2, and CYP3A4) and SXR response element-luciferase activity were increased by bisphenol A treatment in normal osteoblasts transfected with SXR (hFOB/SXR) and in osteoblast-like cells (MG-63). Bisphenol A also stimulated cell proliferation and collagen accumulation in hFOB/SXR cells. These results suggest that, as in other tissues, SXR plays important roles in bone metabolism and fetal bone development and that bisphenol A may disturb bone homeostasis in both adult and fetus through SXR.

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.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.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 (p0.0001) and increased proliferation of ERβ overexpressing A549 cells (p0.0001). Aromatase inhibitor i.e. letrozole abrogated this proliferation and also enhanced the androgenic activity (p0.0001). Testosterone treatment resulted in estrogen responsive elements activation (p0.0001) in ERβ vector transfected A549 and LK87 cells whereas ER blocker i.e. fulvestrant abrogated this effect, (p0.0001).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.

What's known on the subject? and What does the study add? Localization of PDE9 in human LUT has not been reported in the literature, although PDE5 was reported to be abundantly present only in smooth muscle cells using immunohistochemistry. This is the first reported study which demonstrated the expression of PDE9 in the urothelium and neuron cells in the intramural ganglia of human LUT. Patterns of PDE9 localization were clearly different from those of PDE5, which suggest that functions of PDE9 were therefore considered to be different from those of PDE5 in human LUT. OBJECTIVES • To examine the roles of the one of the cyclic guanosine monophosphate (cGMP)-specific phosphodiesterases (PDEs), PDE9, in human lower urinary tract (LUT), we performed immunohistochemistry (IHC) using autopsy specimens. • To demonstrate the potential functional differences between PDE5 and PDE9 in human LUT, the localization of PDE5 and PDE9 was also compared using laser-capture microdissection (LCM)/reverse transcriptase-polymerase chain reaction (RT-PCR) method. MATERIALS AND METHODS • We immunolocalized PDE9 in human bladder (60 cases) and prostate (40) specimens using IHC. • We performed LCM/RT-PCR evaluation in six human bladders to determine the differential expression patterns of PDE5 and PDE9. RESULTS • PDE9 immunoreactivity was detected in the bladder urothelium of all the cases and in 85% of the prostatic urethral urothelium. • PDE9 immunoreactivity was detected in the perikaryon of the intramural ganglia. • LCM/RT-PCR evaluation showed that PDE5 mRNA was exclusively detected in smooth muscle cells but PDE9 mRNA was mainly detected in the urothelium of the human bladder. CONCLUSION • PDE9 is widely distributed in the urothelial epithelium of the human LUT and its potential roles may be different from those of PDE5.

Nudix-type motif 2 (NUDT2) hydrolyzes diadenosine 5',5'''-p1,p4-tetraphosphate (Ap4A) associated with various cellular functions. Previous studies demonstrated its regulation through estrogens, suggesting possible importance of NUDT2 in breast carcinoma. NUDT2, however, has not been examined in malignant tissues. Therefore, we examined its expression and functions in breast carcinoma. Immunohistochemistry for NUDT2 was examined by invasive ductal carcinoma (IDC: n = 145) and pure ductal carcinoma in situ (DCIS: n = 82), and NUDT2 mRNA was examined by real-time PCR in 9 DCIS, 19 IDC and 6 non-neoplastic breast tissues. We also used T47D breast carcinoma cells in in vitro studies. NUDT2 immunoreactivity was detected in 78% of DCIS and 63% of IDC, and NUDT2 mRNA level was significantly higher in DCIS or IDC than non-neoplastic breast. NUDT2 status was significantly correlated with Van Nuys classification, HER2 or Ki-67 in DCIS, and with stage, lymph node metastasis, histological grade or HER2 in IDC. NUDT2 status was significantly associated with adverse clinical outcome of IDC patients and proved an independent prognostic factor. Results of transfection experiments demonstrated that proliferation activity of T47D cells was significantly associated with NUDT2 expression level according to the treatment of estradiol and/or tamoxifen. NUDT2 expression was significantly decreased by estradiol, and it was also significantly decreased in T47D cells transfected with HER2 siRNA. These findings suggest that NUDT2 is an estrogen-repressed gene and is also induced by HER2 pathways in breast carcinoma cells. NUDT2 promotes proliferation of breast carcinoma cells and is a potent prognostic factor in human breast carcinomas.

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 5α-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 17β-hydroxysteroid dehydrogenase type 2 (17βHSD2) status was significantly altered to be positive after the treatment. Following in vitro studies showed that 17βHSD2 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 17βHSD2 expression was markedly suppressed by estradiol (E2) in T-47D cells. E2-mediated cell proliferation was significantly inhibited by DHT in T-47D cells, associated with an increment of 17βHSD2 expression level. These findings suggest that intratumoral androgen actions are increased during exemestane treatment. 17βHSD2 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.

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 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) are involved in the production of estrogens. The reaction related to 17beta-HSDs activity is one of the last steps of estradiol biosynthesis, and 14 isozymes of 17beta-HSD have been identified at this juncture. The balance of the relative expression levels of 17beta-HSD isozymes in human breast carcinomas is thought to play a pivotal role in supply of estradiol to estrogen receptor positive carcinoma cells. Understanding the character of 17beta-HSD isozymes in human breast carcinoma thus provides important information on the mechanisms of biosynthesis of estradiol in breast carcinoma and for development of a therapeutic agent targeted for inhibition of local estradiol synthesis in breast carcinoma cells. In the present review we summarize the roles played by 17beta-HSDs in human breast carcinoma to obtain a better understanding of the properties of 17beta-HSDs in human breast carcinoma.

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-beta and PR were detected in the great majority of the cases (23 and 24 cases, respectively) but ER-alpha 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-beta 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 5 alpha-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.

Aromatase inhibitors have become the gold standard of endocrine therapy in postmenopausal patients with estrogen receptor-positive or estrogen-dependent breast carcinoma, replacing tamoxifen. However, it is true that there are some potential problems to be overcome or improved on regarding aromatase inhibitor treatment of breast cancer. This especially includes the presence of the estrogen receptor-positive patients who do not necessarily respond to aromatase inhibitors, may require other modes of endocrine therapy and develop resistance to aromatase inhibitor in their clinical course, and who may also need alternative modes of suppressing intratumoral estrogen signals or other intracellular signal pathways related to tumor progression or development. Intratumoral estrogen production from precursors present in circulation in an 'intracrine' manner is considered to play very important roles in the development and progression of estrogen receptor-positive breast cancer. The great majority of estrone in circulation is present as a sulfated form or estrone sulfate, and steroid sulfatase hydrolyzes circulating estrone sulfate to estrone in various human tissues in situ, which confers potent estrogenic actions. Estrone is subsequently reduced to 17β-estradiol by 17β-hydroxysteroid dehydrogenase type 1. Therefore, these two enzymes also play very important roles in intracrinology of estrogen in breast cancer in addition to intratumoral aromatase, and the potential inhibition of these two enzymes could lead to the development of a new mode of endocrine therapy based on intracrinology, which may overcome some of the problems above in aromatase inhibitor therapy. In this review, the potential advantages and pitfalls or problems associated with the inhibition of these two intratumoral enzymes in breast cancer patients will be discussed.

Several models of UAVs have been developed for scientific missions such as aerial geomagnetic survey, meteorological observation, and ground observation instead of manned airplanes especially in Antarctica. The UAV fleet is divided into three groups, i.e., a small UAV for low altitude, relatively large UAVs for mid-altitude, and a small glider UAV for high altitude. Several results, operational experiences of the UAVs, lessons learned including the operations in Antarctica, and several problems to be solved are reported in this chapter.

Gastrin-releasing peptide (GRP) belongs to the family of bombesin-like peptides. GRP was demonstrated to stimulate the proliferation and invasiveness of androgen-independent prostate carcinoma. GRP mediates its action through the membrane-bound receptor, GRP receptor (GRPR), which is characterized by a high-affinity binding for both GRP and bombesin. In human prostate cancer tissue, GRPR mRNA was reported to be detectable in more than 90% but its immunolocalizaition has not been reported. Therefore, in this study we immunolocalized GRPR in 51 human prostate cancer cases and correlated the findings with several clinicopathological parameters in order to better understand the function and regulation of GRPR in human prostate cancer. GRPR was immnolocalized in carcinoma cells and their values were significantly associated with Gleason score and immunoreactivity of estrogen receptor βcx (ERβcx) that is one of splicing variants of ligand dependent transcription factor, ERβ, and considered to be prognostic factor of prostate cancer patients. The amounts of GRPR and ERβcx mRNA in three prostate cancer cell lines PC-3, DU-145 and LNCaP evaluated by quantitative RT-PCR (qPCR) analysis were also significantly correlated. In addition, we established stable transformants of prostate carcinoma cell line PC-3 introduced with ERβcx, and confirmed that GRPR mRNA was induced in ERβcx over-expressing PC-3 cells by qPCR analysis. These results also suggest that ERβcx contributes to prostate cancer development possibly through mediating GRPR expression in carcinoma cells.

To examine the roles of the one of the cyclic guanosine monophosphate (cGMP)-specific phosphodiesterases (PDEs), PDE9, in human lower urinary tract (LUT), we performed immunohistochemistry (IHC) using autopsy specimens. To demonstrate the potential functional differences between PDE5 and PDE9 in human LUT, the localization of PDE5 and PDE9 was also compared using laser-capture microdissection (LCM)/reverse transcriptase-polymerase chain reaction (RT-PCR) method.• We immunolocalized PDE9 in human bladder (60 cases) and prostate (40) specimens using IHC. We performed LCM/RT-PCR evaluation in six human bladders to determine the differential expression patterns of PDE5 and PDE9.PDE9 immunoreactivity was detected in the bladder urothelium of all the cases and in 85% of the prostatic urethral urothelium. PDE9 immunoreactivity was detected in the perikaryon of the intramural ganglia. LCM/RT-PCR evaluation showed that PDE5 mRNA was exclusively detected in smooth muscle cells but PDE9 mRNA was mainly detected in the urothelium of the human bladder.PDE9 is widely distributed in the urothelial epithelium of the human LUT and its potential roles may be different from those of PDE5.

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-alpha 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.

17beta-hydroxysteroid dehydrogenase type 12 (17beta-HSD12) has been demonstrated to be involved in enzymatic conversion of weak estrogen, estrone to more potent one, estradiol. However, this enzyme was also reported to be involved in an elongation of very long chain fatty acid (VLCFA). Many genes involved in lipid metabolism are regulated by the transcription factor termed sterol regulatory element-binding proteins (SREBPs). Results of our present study demonstrated that the existence of putative SRE sequence which is recognized as responsive element for SREBPs in 5'-flanking region of 17beta-HSD12 gene. Results of luciferase assay demonstrated that the transcriptional activity of this SRE sequence depends on the activation of SREBP-1 in HepG2 (hepatocellular carcinoma cell line, human) and SK-BR-3 (breast carcinoma cell line, human). 17beta-HSD12 expression was also induced in the HepG2 cells treated with the absence of sterols in which SREBPs were activated. All these results obtained in this study clearly indicate that SREBP-1 represents one of the transcriptional regulators of human 17beta-HSD12.

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 17beta-hydroxysteroid dehydrogenases (17beta-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.

17β-Hydroxysteroid dehydrogenase type 12 (17β-HSD12) has been shown to be involved in elongation of very long chain fatty acid (VLCFA) as well as in biosynthesis of estradiol (E2). 17β-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 17β-HSD12 expression and E2 concentration. We then immunolocalized this enzyme in 110 cases of invasive ductal carcinoma. 17β-HSD12 immunoreactivity in breast carcinoma cells was significantly associated with poor prognosis of the patients. We further examined the biological significance of 17β-HSD12 using cell-based studies. Small interfering RNA–mediated knockdown of 17β-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 17β-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 17β-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. [Cancer Res 2009;69(4):1392–9]

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 alpha. 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 alpha 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.

Previous epidemiologic and in vitro studies have indicated a potential involvement of estrogens in the pathogenesis of human colon carcinoma, but the precise roles of estrogens have remained largely unknown. Therefore, in this study, we first measured intratumoral concentrations of estrogens in 53 colon carcinomas using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS). Tissue concentrations of total estrogen [estrone (E1) + estradiol] and E1 were significantly (2.0- and 2.4-fold, respectively) higher in colon carcinoma tissues than in nonneoplastic colonic mucosa (n = 31), and higher intratumoral concentrations of total estrogen and E1 were significantly associated with adverse clinical outcome. Intratumoral concentration of total estrogen was significantly associated with the combined status of steroid sulfatase (STS) and estrogen sulfotransferase (EST), but not with that of aromatase. Thus, we subsequently examined the STS/EST status in 328 colon carcinomas using immunohistochemistry. Immunoreactivities for STS and EST were detected in 61% and 44% of the cases, respectively. The −/+ group of the STS/EST status was inversely associated with Dukes' stage, depth of invasion, lymph node metastasis, and distant metastasis and positively correlated with Ki-67 labeling index of the carcinomas. In addition, this −/+ group had significantly longer survival, and a multivariate analysis revealed the STS/EST status as an independent prognostic factor. Results from our present study showed that the STS/EST status of carcinoma tissue determined intratumoral estrogen levels and could be a significant prognostic factor in colon carcinoma, suggesting that estrogens are locally produced mainly through the sulfatase pathway and play important roles in the progression of the disease. [Cancer Res 2009;69(3):914–22]

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.

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.

This paper presents an automatic design method of UAV configurations using EA (Evolutionary Algorithm). Aerodynamic configuration as well as the design variables which include the parameters necessary for sizing are optimized. In automatic generation of configuration, the four principal components are defined and the combination of them are randomly selected. The design variables are optimized evaluating an objective function which reflects design requirements and the limits. Some examples show that the possibility to design a unique UAV which cannot be designed easily by human designers.