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) a (A549 + ERa) or ERh (A549 + ERh) 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 ERa-or ERh-positive cases. In in vitro studies, estradiol significantly increased cell proliferation of A549 + ERa orA549 + ERh, which was significantly suppressed by selective ER modulators, tamoxifen or raloxifene. Both A549 + ERa and A549 + ERh 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 ERa-or ERh-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.

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-a 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 intratumoral 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.

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 5a-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 (nZ9) than those without the therapy (nZ7), and 17b-hydroxysteroid dehydrogenase type 2 (17bHSD2) status was significantly altered to be positive after the treatment. Following in vitro studies showed that 17bHSD2 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 17bHSD2 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 17bHSD2 expression level. These findings suggest that intratumoral androgen actions are increased during exemestane treatment. 17bHSD2 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.

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 (E(1)) + estradiol] and E(1) 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 E(1) 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.

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 estrogenindependent transcriptional activity of estrogen receptor a. 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. (1) They belong to the steroid and thyroid hormone superfamily of nuclear receptors and are involved in regulating the expression of various genes.(1) COUP-TF were initially characterized in chick oviduct (2) and HeLa extracts, (3) where they bind as dimers to the COUP element of the ovalbumin gene promoter to activate transcription.(2) COUP-TF were therefore initially characterized as transcriptional activators of the chicken ovalbumin gene but are currently also considered as repressors of the transcriptional activity of other nuclear hormone receptors (4) such as retinoic acid receptor, thyroid hormone receptor, peroxisome proliferatoractivated receptor, and vitamin D receptor, via direct interaction, competing for their DNA binding sites, or by heterodimerization with retinoid X receptor.(5,6) There are two COUP-TF genes reported in mammals, COUP-TFI and COUP-TFII. These two COUP-TF genes are closely correlated, with an overall amino acid identity of 87%. They are evolutionarily conserved in the DNA binding domain as well as the ligand-binding domain, which suggests that they are relatively primordial members of the nuclear receptor family and have important biological functions. In human breast cancer, the expression of COUP-TFI was reported to be increased in carcinoma cells compared with normal cells and to promote estrogen-independent transcriptional activity of ERα. (8,9) Therefore, the expression of COUP-TFI in ERα-positive breast cancer is reasonably postulated to be involved in its progression. In addition, high amounts of COUP-TFII expression were reported in invasive lung carcinoma cell lines (10) and in dedifferentiated endometrial (11) and ovarian cancer cell lines.(12) All of these reporteds findings suggest that COUP-TFII expression may affect breast cancer cell progression through modulation of other nuclear receptors such as ERα, as well as COUP-TFI, or through other pathways. However, the clinical significance of COUP-TFII in breast carcinoma remains virtually unknown.Therefore, in the present study, we examined the immunolocalization of COU-TFII in 119 cases of human breast carcinoma, and correlated these findings with various clinicopathological parameters in order to clarify the biological and clinical significance of this orphan nuclear receptor in human breast cancer progression. In addition, we examined the potential regulation of VEGF-C mRNA expr...