Increased Aromatase Expression Research Articles

Prevention and treatment of breast cancer by suppressing aromatase activity and expression.

Estrogen promotes the proliferation of breast cancer cells. Aromatase is the enzyme that converts androgen to estrogen. In tumors, expression of aromatase is upregulated compared to that of surrounding noncancerous tissue. Tumor aromatase is thought to stimulate breast cancer growth in both an autocrine and a paracrine manner. A treatment strategy for breast cancer is to abolish in situ estrogen formation with aromatase inhibitors. In addition, aromatase suppression in postmenapausal women is being evaluated as a potential chemopreventive modality against breast cancer. One area of aromatase research in this laboratory is the identification of foods and dietary compounds that can suppress aromatase activity. In vitro and in vivo studies have found that grapes and mushrooms contain chemicals that can inhibit aromatase. Therefore, a diet that includes grapes and mushrooms would be considered preventative against breast cancer. Another area of our aromatase research is the elucidation of the regulatory mechanism of aromatase expression in breast cancer tissue. Increased aromatase expression in breast tumors is attributed to changes in the transcriptional control of aromatase expression. Whereas promoter I.4 is the main promoter that controls aromatase expression in noncancerous breast tissue, promoters II and I.3 are the dominant promoters that drive aromatase expression in breast cancer tissue. Our recent gene regulation studies revealed that in cancerous versus normal tissue, several positive regulatory proteins (e.g., nuclear receptors and CREB1) are present at higher levels and several negative regulatory proteins (e.g., snail and slug proteins) are present at lower levels. This may explain why the activity of promoters II and I.3 is upregulated in cancerous tissue. In addition, our in vitro transcription/translation analysis using plasmids containing T7 promoter and the human snail gene as a reporter capped with different untranslated exon Is revealed that exon PII-containing transcripts were translated more effectively than were exon I.3-containing transcripts. An understanding of the molecular mechanisms of aromatase expression between noncancerous and cancerous breast tissue, at both transcriptional and translational levels, may help in the design of a therapy based on suppressing aromatase expression in breast cancer tissue.

Expression of aromatase P450 is increased in spontaneous prolactinomas of aged rats.

We have recently reported the presence of aromatase P450 in the rat hypophysis. This enzyme is responsible for the aromatization of testosterone to estradiol. Since the induction of prolactinomas has been demonstrated in the rat following chronic treatment with estradiol, the aim of the present study was to analyze whether a relationship exists between the presence of pituitary aromatase and the appearance of spontaneous prolactinomas in aged rats. Of a series of 90 adenomas studied, 53% showed prolactin immunoreactive cells and were classified as prolactinomas; only 33% of the adenomas were pure prolactinomas and the other 20% were multi-hormonal protactinomas. Moreover, 60% of the adenomas were aromatase-positive tumors. Interestingly, 100% of the pure prolactinomas were aromatase-positive while only 60% of the multi-hormonal prolactinomas expressed the enzyme. Western blotting with anti-aromatase antibodies revealed a 3.8-fold increase in expression of aromatase in pituitary tumors as compared to normal rat pituitary gland. Double immunohistochemical labeling detected the coexistence of prolactin and aromatase P450 in prolactinoma cells. ACTH- and LH-positive adenomas were considered as controls; only multi-hormonal ACTH and LH tumors display aromatase-positive cells and all of these also contained prolactin-positive cells. Our results demonstrate for the first time that aromatase is expressed in pituitary adenomas and that it is related to the functional nature of the tumor, especially in the case of pure prolactinomas, suggesting the possibility that an abnormally high conversion of testosterone into estradiol in pituitary cells may contribute to the genesis of spontaneous prolactinomas in aged rats.

Aromatase expression in the human male

The role of estrogens produced by the testis may involve negative feedback regulation of androgen biosynthesis. Estrogens are also associated with contractile processes of seminiferous tubules, and might have mitogenic effects on Sertoli and Leydig cells. To investigate the location of aromatase (estrogen synthetase) in the testes, tissue from normal human subjects, aged 3 months to 72 years were studied using immunocytochemistry. In mature testes, aromatase immunostain was always associated with Leydig cells and was absent from Sertoli cells. Aromatase activity ranged from 0.014–0.55 pmol estrogen per mg/h and was significantly correlated with the immunostain intensity ( P<0.02). Activity and immunostain intensity did not correlate with increasing age. Rather, the highest levels were measured in four of six testes of men aged 18–20 years, three of whom also had the strongest immunostain in larger and more prominent Leydig cell clusters than those in the other specimens. A low level of aromatase activity but no immunostain was detected in prepubertal testes. However, in several prepubertal patients with Peutz–Jegher's Syndrome (PJS) with bilateral multifocal sex cord tumors and enlarged seminiferous tubules and Sertoli cells, aromatase was expressed in these Sertoli cells, but absent from normal Sertoli and Leydig cells. Increased aromatase expression in these tissues involved activation of upstream regulatory elements of the gonadal P II promoter of P-450 arom. In a prepubertal boy with gynecomastia but without PJS, aromatase excess appeared to be due to increased aromatization in skin fibroblasts and lymphocytes. Several members of the patient's family including his sister also expressed high levels of aromatase. This condition appears to be inherited in an autosomal dominant manner.

Expression of 3β-hydroxysteroid dehydrogenase, cytochrome P450 17α-hydroxylase/17,20-lyase and cytochrome P450 aromatase enzymes in corpora lutea of diestrous and early pregnant mares

In the pregnant mare, luteal estrogen production increases at the onset of equine chorionic gonadotropin (eCG) secretion by endometrial cups. In previous studies, we have demonstrated that eCG stimulates luteal androgen and estrogen production in pregnant mares. To further elucidate the regulation of steroidogenesis within the equine corpus luteum (CL) of pregnancy, we examined the expression of 3β-hydroxysteroid dehydrogenase (3β-HSD), cytochrome P450 17α-hydroxylase/17,20 lyase (P450 17α) and cytochrome P450 aromatase (P450 arom) in luteal tissue samples collected during diestrus (Days 7 to 10) and pregnancy before (Days 29 to 35) and after (Days 42 to 45) the onset of eCG secretion. Immunoblot analyses revealed a single protein per enzyme with molecular weights of 48 kDa (3β-HSD), 58 kDa (P450 17α) and 56 kDa (P450 arom). Steady-state levels of 3β-HSD were lower in luteal tissue of diestrus than pregnancy, but expression did not change during pregnancy. Steady-state expression of P450 17α in CL of diestrus was not significantly different from that of pregnancy. During pregnancy, P450 17α expression was significantly higher after the onset of eCG secretion. Steady-state expression of P450 arom in CL of diestrus was not significantly different from that of pregnancy. During pregnancy, luteal expression of P450 arom was significantly lower after the onset of eCG secretion. These data support the hypotheses that eCG has a differential effect on the expression of luteal steroidogenic enzymes, that the eCG-induced increase in luteal estrogen production is the result of an increase in available aromatizable androgen due to an increase in P450 17α expression and activity, and that increased luteal estrogen production is not due to an increase in aromatase expression.

Immunolocalization of aromatase in human minor salivary glands of the lower lip with primary Sjögren's syndrome.

The enzyme aromatase is involved in the conversion of androgens to estrogens and in the modulation of various androgenic and estrogenic actions. Abnormalities of estrogen metabolism have been postulated to play roles in the development and/or pathophysiology of Sjögren's syndrome. In the present study, aromatase was immunolocalized in 75 cases of inflammatory disorders of human minor salivary glands of the lower lip. These included cases of primary Sjögren's syndrome (19 cases), of chronic sialadenitis (34 cases) and of mucous extravasation cysts (22 cases), in order to clarify the possible involvement of in situ estrogen production in primary Sjögren's syndrome. Aromatase immunoreactivity was detected in myoepithelial cells of acini and in interstitial cells adjacent to acini and ducts in 13/19 (68%) cases of primary Sjögren's syndrome. In contrast, aromatase expression was detected in only six of 34 (18%) cases of chronic sialadenitis and in seven of 22 (32%) cases of mucous extravasation cyst. These results suggest that increased aromatase expression in minor salivary glands with primary Sjögren's syndrome in premenopausal women may be involved in the biological features of primary Sjögren's syndrome through the production of estrogens in situ and possibly through the aggravation of the inflammatory reaction.

Aromatase cytochrome P450 gene expression in endometrial carcinoma.

We analysed aromatase gene expression and its regulation in seven cases of endometrioid endometrial carcinoma. Immunohistochemistry revealed the presence of strong aromatase immunoreactivity in the stromal cells of carcinoma in five out of seven cases. A polymerase chain reaction after reverse transcription (RT PCR) revealed varying levels of aromatase transcripts (0.1-27.0 amol ng(-1) total mRNA) in five cases. The alternative use of multiple exons 1 was also examined by identifying various human aromatase transcripts specific for exons 1 in RT-PCR products. Gonadal type or exon 1d was primarily used in three cases in which aromatase overexpression was not detected. The two cases in which fibroblasts type or exon 1b was used with other exons 1 as minor transcripts demonstrated aromatase overexpression in immunohistochemistry and RT PCR analysis. Further studies are required, but alternative splicing as well as use of multiple exons 1 transcripts may result in increased aromatase expression in stromal cells observed in endometrial carcinoma.

Aromatase and steroid receptors in gynecomastia and male breast carcinoma: an immunohistochemical study.

Hormonal factors have been implicated in the development of male breast disorders, including carcinoma and gynecomastia. We studied the expression of aromatase and estrogen (ER), progesterone (PR), and androgen (AR) receptors by immunohistochemistry in male breast carcinoma (15 cases) and gynecomastia (30 cases) to evaluate their possible significance in these disorders. Relatively strong aromatase immunoreactivity was observed in all cases of carcinoma, but in only 11 of 30 cases (37%) of gynecomastia. ER and PR expression was observed in the nuclei of ductal cells in all the cases of gynecomastia. More than 10% of the carcinoma cells were positive for ER and PR in 9 of 15 (60%) and 10 of 15 (67%) carcinomas, respectively. AR immunoreactivity was observed in nuclei of both epithelial and non-epithelial cells. AR was present in ductal or carcinoma cells in 13 of 15 (87%) cases of carcinoma and in all 30 (100%) cases of gynecomastia. The mean percentage of ER-, PR-, and AR-positive cells were significantly higher in gynecomastia than in carcinoma. There was a close association of AR with ER (P < 0.01) and PR (P < 0.01) in cases of gynecomastia, but there was a significant inverse correlation between AR and ER (P < 0.01) or PR (P < 0.05) expression in carcinoma cases. Increased aromatase expression in the stromal cells is considered to contribute to the increment in the in situ estrogen concentration and the development of male breast carcinoma.

Cellular characterization of adipose tissue from various body sites of women

Adipose tissue is the primary site of estrogen biosynthesis in postmenopausal women. The two main histologic components of adipose tissue are mature adipocytes and fibroblasts. Aromatase P450 expressed in the fibroblast component of adipose tissue is responsible for catalyzing conversion of C19 steroids to estrogens. We previously have demonstrated that in women, aromatase expression in adipose tissue of various body sites increases with age and that aromatase expression in the hip is markedly higher than in the abdomen. To determine whether this age- and regional-dependent variation in aromatase expression is caused by an alteration in the ratio of fibroblasts to mature adipocytes, we collected sc adipose tissue samples from 19 women (age range: 21-93 yr) at the time of autopsy. Using a computerized image analysis system, we determined by morphometry the proportions of adipocytes, fibroblasts, and vascular endothelial cells within histologic sections of adipose tissue from midabdomen, both breasts, and both hips. The percentage of each cell component at each body site was expressed as the mean of triplicate replicates. Statistical analysis of our results did not indicate any correlation between advancing age and fibroblast to adipocyte ratios in the breast, abdomen, or hip. Fibroblast to adipocyte ratios were found to be significantly higher in the breast and abdomen compared with the hip ( P < 0.05). No statistical differences were found between the breast and abdomen. These findings suggest that the increase in aromatase expression with advancing age and the higher aromatase expression in the hip compared with the abdomen in women may be caused by alterations in specific signal transduction mechanisms rather than a simple increase in local adipose fibroblast numbers.

Use of tissue-specific promoters in the regulation of aromatase cytochrome P450 gene expression in human testicular and ovarian sex cord tumors, as well as in normal fetal and adult gonads.

We have previously demonstrated that the tissue-specific regulation of human aromatase cytochrome P450 (P450arom) gene expression is, in part, the consequence of the use of tissue-specific promoters. Promoter I.1 (PI.1) and PI.2-specific transcripts are expressed in the placenta, whereas promoter II (PII) appears to be the only active promoter in the corpus luteum. Testicular and ovarian sex cord tumors with annular tubules (SCTATs) associated with gynecomastia in prepubertal boys and isosexual precocity in girls with Peutz-Jeghers syndrome (P-JS) have been previously reported. In the present study, we investigated the regulatory elements directing P450arom gene transcription in samples of SCTAT from three prepubertal boys and a girl with P-JS and an ovarian granulosa cell tumor from an adult woman, as well as in healthy fetal and adult testicular and ovarian tissues. Placental tissue was used as a control. Using polymerase chain reaction linked to reverse transcription and northern blotting, we determined the tissue-specific use of various P450arom promoters by analyzing specific 5'-termini from messenger RNA templates. Results indicate a universal gonadal promoter (PII) directs P450arom gene expression in healthy fetal and adult ovaries and testes, as well as in SCTAT of the P-JS and an adult ovarian granulosa cell tumor. These results are interpreted to mean that use of PII in human ovary and testis is preserved from the fetal period into adult life as well as in transformed neoplastic Sertoli and granulosa cells. On the other hand, transcripts from placenta are specific for PI.1 (and to a much lesser extent, PI.2). In SCTAT, immunoreactive P450arom is detected only in the cytoplasm of neoplastic cells, whereas the normal-appearing sex cords do not contain any immunoreactive P450arom. These results further suggest that the markedly increased aromatase expression of these transformed neoplastic cells is not a consequence of using different tissue-specific promoters. Rather it appears to involve activation (or failure of inhibition) of the upstream regulatory elements of the same promoter, which is normally functional in all gonadal tissues, namely the proximal PII.