Mitsutoshi Tamura

Regulation of Aromatase Expression in Estrogen-Responsive Breast and Uterine Disease: From Bench to Treatment

A single gene encodes the key enzyme for estrogen biosynthesis termed aromatase, inhibition of which effectively eliminates estrogen production. Aromatase inhibitors successfully treat breast cancer and endometriosis, whereas their roles in endometrial cancer, uterine fibroids, and aromatase excess syndrome are less clear. Ovary, testis, adipose tissue, skin, hypothalamus, and placenta express aromatase normally, whereas breast and endometrial cancers, endometriosis, and uterine fibroids overexpress aromatase and produce local estrogen that exerts paracrine and intracrine effects. Tissue-specific promoters distributed over a 93-kilobase regulatory region upstream of a common coding region alternatively control aromatase expression. A distinct set of transcription factors regulates each promoter in a signaling pathway- and tissue-specific manner. Three mechanisms are responsible for aromatase overexpression in a pathologic tissue versus its normal counterpart. First, cellular composition is altered to increase aromatase-expressing cell types that use distinct promoters (breast cancer). Second, molecular alterations in stromal cells favor binding of transcriptional enhancers versus inhibitors to a normally quiescent aromatase promoter and initiate transcription (breast/endometrial cancer, endometriosis, and uterine fibroids). Third, heterozygous mutations, which cause the aromatase coding region to lie adjacent to constitutively active cryptic promoters that normally transcribe other genes, result in excessive estrogen formation owing to the overexpression of aromatase in many tissues.

Role of aromatase in endometrial disease.

Aromatase is the key enzyme for estrogen biosynthesis. It is normally expressed in the human ovary, skin, adipose tissue and brain. Aromatase activity is not detectable in normal endometrium. In contrast, aromatase is expressed aberrantly in endometriosis and is stimulated by PGE2. This results in local production of estrogen, which induces PGE2 formation and establishes a positive feedback cycle. Another abnormality in endometriosis, i.e. deficient 17beta-hydroxysteroid dehydrogenase (17beta-HSD) type 2 expression, impairs the inactivation of estradiol to estrone. These molecular aberrations collectively favor accumulation of increasing quantities of estradiol and PGE2 in endometriosis. The clinical relevance of these findings was exemplified by the successful treatment of an unusually aggressive case of post-menopausal endometriosis using an aromatase inhibitor.

Aromatase in endometriosis and uterine leiomyomata

Endometrial tissue from uterine disease-free women does not exhibit aromatase activity. In contrast, aromatase enzyme activity and mRNA levels are readily detectable in endometriosis. PGE2 stimulates both aromatase expression and activity in endometriotic stromal cells via promoter II region of the aromatase gene. This results in local production of estradiol, which induces PGE2 formation and establishes a positive feedback cycle. This mechanism seems to contribute to continuous production of estradiol and PGE2. Aromatase mRNA levels and enzyme activity are also present in uterine leiomyomata that are estrogen-dependent benign tumors of the myometrium. Successful treatment of endometriosis and uterine leiomyomata using aromatase inhibitors by recent pilot trials underscores the clinical significance of these molecular studies.

Estrogen Production and Metabolism in Endometriosis

Abstract: Aromatase activity is absent in normal endometrium. In contrast, aromatase is expressed aberrantly in endometriosis, which gives rise to strikingly high levels of aromatase activity in this tissue. Both aromatase expression and activity are stimulated by PGE2. This results in local production of estrogen, which induces PGE2 formation and establishes a positive feedback cycle. Another abnormality in endometriosis, that is, deficient 17β‐HSD type 2 expression, impairs the inactivation of estradiol to estrone. These molecular aberrations collectively favor accumulation of increasing quantities of estradiol and PGE2 in endometriosis. The clinical relevance of these findings was exemplified by the successful treatment of an unusually aggressive case of postmenopausal endometriosis using an aromatase inhibitor.

Mechanisms of excessive estrogen formation in endometriosis

Estrogen is produced in a number of human tissues including the ovary, placenta and extraglandular sites such as adipose tissue, skin and the brain. Aromatase is the key enzyme that regulates estrogen formation in these tissues. Aromatase activity is not detectable in normal endometrium. In contrast, aromatase is expressed aberrantly in endometriosis and is stimulated by PGE(2). This results in local production of estrogen, which induces PGE(2) formation and establishes a positive feedback cycle. Another abnormality in endometriosis, i.e. deficient 17beta-hydroxysteroid dehydrogenase (17beta-HSD) type 2 expression, impairs the inactivation of estradiol to estrone. These molecular aberrations collectively favor accumulation of increasing quantities of estradiol and PGE(2) in endometriosis. The clinical relevance of these findings was exemplified by the successful treatment of an unusually aggressive case of postmenopausal endometriosis using an aromatase inhibitor.

Safe techniques in surgery for hysteroscopic myomectomy

Hysteroscopic myomectomy is regarded as the best treatment for patients with submucous myomata. However, this procedure has a number of associated complications, including uterine perforation, cervical laceration, hyponatremia and hemorrhage, especially in cases of sessile submucous myomata. To avoid these problems, it is important to make well‐advised preparations and manipulations both pre‐ and intraoperatively. The main surgical considerations for safe hysteroscopic myomectomy are shortening the operating time and avoiding cutting too deeply into the myometrium. With these requirements in mind, a combination of techniques using vaporization and a powerful oxytocic agent, such as prostaglandin F‐2α, appears to be the safest method of carrying out hysteroresectoscopy for unpedunculated sessile submucous myomata.

X-chromosome inactivation in the human trophoblast of early pregnancy

AbstractTo investigate X-chromosome inactivation (XCI) in human trophoblasts during early pregnancy, tropho-blast genomic DNA was extracted and analyzed for a Bst XI restriction endonuclease site polymorphism in the X-linked phosphoglycerate kinase gene, after digestion with methylation-sensitive Hpa II (control samples were digested instead with Afa I). Six villous trophoblast DNA samples were informative for the polymorphism (ie, heterozygous) and were derived from women homozygous for the polymorphism. These samples were then evaluated for XCI. In five of the six samples with Hpa II predigestion, the sizes of the two heterozygous band peaks differed; maternal X-chromosome (XM)-derived alleles showed smaller peak sizes than paternal X-chromosome (XP)-derived alleles, but the differences varied in degree. In samples obtained by microdissection from formalin-fixed, paraffin-embedded tissues (30 samples from different anchoring villi, and 38 samples from different branch villi), monoclonal band patterns of XP-derived alleles were observed more frequently than those of XM-derived alleles, but almost half of the samples showed polyclonal pat-terns. Our results suggest that a skewing of XCI exists in the human trophoblast; however, the degree of non-randomness due to predominant XP inactivation appears to be restricted. It is probable that transcription of the X inactivation center (XIC) begins earlier in mice than in humans.

Peroxisome Proliferator-Activated Receptor γ and Growth Inhibition by Its Ligands in Uterine Endometrial Carcinoma

Purpose: In this study, we evaluated the correlation between endometrial carcinoma and peroxisome proliferator-activated receptor γ (PPARγ) expression and assessed whether PPARγ ligands influence carcinoma growth. Experimental Design: We examined the presence and cellular distribution of PPARγ protein in 42 normal endometria, 32 endometria with hyperplasia, and 103 endometria with endometrial carcinoma by immunohistochemistry. We then compared PPARγ mRNA expression in endometrial carcinoma with that in normal endometria using real-time reverse transcription-PCR. We subsequently confirmed expression of PPARγ mRNA by real-time reverse transcription-PCR and PPARγ protein by immunoblotting in endometrial carcinoma cell lines (Ishikawa, Sawano, and RL95-2 cells). We further examined the effects of PPARγ agonist 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), a naturally occurring PPARγ ligand, to these endometrial carcinoma cell lines. We also examined the status of apoptosis and p21 mRNA expression of these endometrial carcinoma cell lines following addition of 15d-PGJ2. Results: PPARγ immunoreactivity was detected in 11 of 23 (48%) of proliferative-phase endometrium, 14 of 19 (74%) of secretory-phase endometrium, 27 of 32 (84%) of endometrial hyperplasia, and 67 of 103 (65%) of carcinoma cases. PPARγ immunoreactivity was significantly lower in endometrial carcinoma than in secretory-phase endometrium (P = 0.012) and endometrial hyperplasia (P = 0.006). There was a significant positive association between the status of PPARγ and p21 expression in endometrial carcinoma (P < 0.0001). There was a significant negative association between the body mass index and PPARγ labeling index of carcinoma tissue in the patients with endometrial carcinoma (P < 0.0001). PPARγ mRNA was expressed abundantly in normal endometria but not in endometrial carcinoma. We showed that PPARγ agonist 15d-PGJ2 inhibited cell proliferation and induced p21 mRNA of endometrial carcinoma cell lines. Conclusion: We showed the expression of PPARγ in human endometrial carcinoma and the effects of PPARγ ligand in endometrial carcinoma cells. These findings suggest that a PPARγ ligand, 15d-PGJ2, has antiproliferative activity against endometrial carcinoma.

Transforming Growth Factor-β Isoforms and Receptors in Endometriotic Cysts of the Human Ovary

PROBLEM: The present study examined the presence and cellular distribution of transforming growth factor‐β1, 2, and 3 isoforms and their type I and II receptors in endometriotic cysts of the ovary, relative to their presence in normal endometrial tissue.

Complete androgen insensitivity in a 47,XXY patient with uniparental disomy for the X chromosome

We describe a unique patient with complete androgen insensitivity syndrome and a 47,XXY karyotype. Androgen receptor assay using cultured pubic skin fibroblasts showed no androgen-binding capacity. Sequence analysis of the androgen receptor gene demonstrated two nonsense mutations, one in exon D and one in exon E. Microsatellite marker analysis showed that the patient is homozygous for all five Xq loci examined. The results suggest that the long-arms of the two X chromosomes are identical, i.e., uniparental isodisomy at least for Xq, and carry the same mutations in the androgen receptor gene. This explains how complete androgen insensitivity syndrome occurred in this 47,XXY individual.