3ßHSD and CYB5A double positive adrenocortical cells during adrenal development/aging.

Androstenedione is a common precursor of sex steroids produced and secreted in the human adrenal gland and produced by 3ß-hydroxysteroid dehydrogenase (3ßHSD), 17ß-hydroxylase/17,20-lyase (CYP17) and cytochrome b5 (CYB5A). 3ßHSD is expressed in the zona glomerulosa (ZG) and fasciculata (ZF), CYP17 in the ZF and zona reticularis (ZR) and CYB5A in the ZR, respectively. We previously demonstrated the presence of cortical parenchymal cells co-expressing 3ßHSD and CYB5A with hybrid features of both ZF and ZR in human adrenal cortex and hypothesized that these cells may play an important role in androstenedione production in human adrenal gland. Age-related morphologic development of these hybrid cells has, however, not been studied. Therefore, in this study, 48 human adrenal specimens from various age groups were retrieved. Double-immunohistochemical analyses were used in order to study the correlation between this hybrid cell type and age. In both male and female adrenal cortex, the means of total adrenocortical area, the area positive for CYB5A and its ratio reached highest peak in the 21-40-year-old (y.o.) group. The greatest overlap between 3ßHSD and CYB5A in both total and relative area was present in the 13-20 y.o. group. For all the markers mentioned above, statistically significant differences were detected among the different age groups examined (p < 0.05). These findings indicated that both area and ratio of 3ßHSD and CYB5A double positive cells, which could represent the hybrid cells of ZF and ZR, are correlated with human adrenal development and could subsequently influence age-related serum androstenedione levels.

Aromatase inhibitor treatment of breast cancer cells increases the expression of let-7f, a microRNA targeting CYP19A1.

Aromatase inhibitors (AIs) are considered the gold standard of endocrine therapy for oestrogen receptor-positive postmenopausal breast cancer patients. AI treatment was reported to result in marked alterations of genetic profiles in cancer tissues but its detailed molecular mechanisms have not been elucidated. Therefore, we profiled miRNA expression before and after treatment with letrozole in MCF-7 co-cultured with primary breast cancer stromal cells. Letrozole significantly altered the expression profiles of cancer miRNAs in vitro. Among the elevated miRNAs following letrozole treatment, computational analysis identified let-7f, a tumour-suppressor miRNA which targeted the aromatase gene (CYP19A1) expression. Quantitative real-time PCR assay using MCF-7 and SK-BR-3 cells as well as clinical specimens of a neoadjuvant study demonstrated a significant inverse correlation between aromatase mRNA and let-7f expression. In addition, high let-7f expression was significantly correlated with low aromatase protein levels evaluated by both immunohistochemistry and the western blotting method in breast cancer cases. Results of 3'UTR luciferase assay also demonstrated the actual let-7f binding sites in CYP19A1, indicating that let-7f directly targets the aromatase gene. Subsequent WST-8 and migration assays performed in let-7f-transfected MCF-7 and SK-BR-3 cells revealed a significant decrement of their proliferation and migration. These findings all demonstrated that let-7f, a tumour suppressor miRNA in breast cancer, directly targeted the aromatase gene and was restored by AI treatment. Therefore, AIs may exert tumour-suppressing effects upon breast cancer cells by suppressing aromatase gene expression via restoration of let-7f.

Decreased expression of 14-3-3σ is predictive of poor prognosis for patients with human uterine papillary serous carcinoma.

Uterine papillary serous carcinoma (UPSC) morphologically resembles ovarian serous carcinoma and is categorized as a type II endometrial cancer. UPSC comprises about 10% of all types of endometrial cancer and has an aggressive clinical course and a poor prognosis. The 14-3-3σ gene was originally discovered as a p53-inducible gene; its expression is induced by DNA damage in a p53-dependent manner, which leads to G2 arrest and repair of damaged DNA. Moreover, it has been reported that expression of 14-3-3σ is frequently lost in various types of human cancer, including ovarian cancer. We therefore examined the association between 14-3-3σ expression determined by immunohistochemistry and clinical outcomes of 51 patients with UPSC. UPSC was considered positive for 14-3-3σ when > 30% of tumor cells were stained with a specific antibody. Of these patients, 29 (58.7%) showed positive immunoreactivity for 14-3-3σ and 22 (41.3%) had decreased 14-3-3σ staining. Decreased immunoreactivity for 14-3-3σ was associated with stage (P = 0.001) and lymphovascular space involvement (P = 0.005). Moreover, decreased 14-3-3σ expression was an independent risk factor for reduced overall survival (P = 0.0416) in multivariate analysis. Direct bisulfite sequencing was performed to evaluate the methylation status of the 27 CpG islands in the promoter region and first exon of the 14-3-3σ gene. These CpG islands were hypermethylated in 30% of 14-3-3σ-positive UPSC and 80% of 14-3-3σ-negative UPSC, although the difference was not statistically significant. These findings suggest that decreased expression of immunoreactive 14-3-3σ may be a predictor of poor prognosis in patients with UPSC.

Muscular metastasis of superficial esophageal squamous cell carcinoma: a rare case of recurrence after endoscopic submucosal dissection with additional chemoradiotherapy.

Esophageal cancer after endoscopic treatment may recur depending on the risk. We present a case of a rare T1b esophageal cancer after endoscopic treatment plus chemoradiotherapy (CRT) that recurred with metastasis of the dorsal muscles. A 70-year-old man was referred for treatment of early-stage esophageal carcinoma. Endoscopic submucosal dissection (ESD) was performed and histopathology showed a poorly differentiated squamous cell carcinoma with invasion to the submucosal layer (sm2) with INFc-type invasion and positive venous invasion. After subsequent CRT, the patient was monitored every 6 months, using computed tomography (CT) and endoscopy. Fifteen months after the treatment, contrast CT revealed a spherical mass with 9 cm ring enhancement within the right erector spinae, that had squamous cell carcinoma confirmed by CT-guided biopsy. Radiation and systemic chemotherapy were initiated for the metastasis of the esophageal carcinoma. However, he died of respiratory failure due to rapid pleural effusion 26 months after ESD. Pathological autopsy showed diffuse squamous cell carcinoma invasion of the cystic wall, forming a lumbar mass, and absence of cancer cell remnants or recurrences in the esophagus. This case report emphasizes the need for systemic observation of superficial esophageal cancer after treatment with a high risk of recurrence.

MicroRNA-34b functions as a potential tumor suppressor in endometrial serous adenocarcinoma.

Endometrial serous adenocarcinoma (ESC) is aggressive and carries a poor prognosis. p53 is frequently mutated in ESC. microRNAs (miRNAs) are a direct p53 target and have been implicated in cancer cell behavior. In this study, we compared miRNA expression levels in ESC with the levels in endometrial endometrioid adenocarcinoma (EEC) and normal endometria. Six miRNAs were identified as having aberrant down-regulation specific to ESC with miR-34b being most pronounced. miR-34b was found to have promoter hypermethylation, which when reversed, restored miR-34b expression in the cell lines treated with 5-aza-2' deoxycytidine (DAC). Ectopic expression of miR-34b in turn inhibited cell growth, migration and most notably invasion. Our findings suggest a relationship among p53 mutation, miR-34b promoter methylation and tumor cell behavior. These effects are likely mediated by the downstream target of miR-34b, the proto-oncogene mesenchymal-epithelial transition factor (MET), a known prognostic factor in endometrial carcinomas. The expression of MET was reduced following the restoration of miR-34b in cell lines. In summary, our data suggest that miR-34b plays a role in the molecular pathogenesis of endometrial cancer.

Nucleobindin 2 in human breast carcinoma as a potent prognostic factor.

It is well-known that estrogens immensely contribute to the progression of human breast carcinoma, but their detailed molecular mechanisms remain largely unclear. In this study, we identified nucleobindin 2 (NUCB2) as a gene associated with recurrence based on microarray data of estrogen receptor (ER)-positive breast carcinoma cases (n = 10), and subsequent in vitro study showed that NUCB2 expression was upregulated by estradiol in ER-positive MCF-7 cells. However, NUCB2 has not yet been examined in breast carcinoma, and its significance remains unknown. Therefore, we further examined the biological functions of NUCB2 in breast carcinoma using immunohistochemistry and in vitro studies. NUCB2 immunoreactivity was detected in carcinoma cells in 77 of 161 (48%) breast cancer cases, and positively associated with lymph node metastasis and ER status of the patients. In addition, NUCB2 status was significantly associated with an increased risk of recurrence and adverse clinical outcome of the patients using both univariate and multivariate analyses. Results of siRNA transfection experiments showed that NUCB2 significantly increased cell proliferation, and migration and invasion properties in both MCF-7 and ER-negative SK-BR-3 cells. These results suggest that NUCB2 is upregulated by estrogens and plays an important role, especially in the process of metastasis, in breast carcinomas. NUCB2 status is considered a potent prognostic factor in human breast cancer.

Nudix-type motif 2 in human breast carcinoma: a potent prognostic factor associated with cell proliferation.

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.

Novel classification based on immunohistochemistry combined with hierarchical clustering analysis in non-functioning neuroendocrine tumor patients.

Somatostatin analogues ameliorated many symptoms caused by neuroendocrine tumors (NET), but their antitumor activities are limited especially in non-functioning cases. An overactivation of signaling pathways under receptor tyrosine-kinase (RTK) has been recently demonstrated in some NET patients, but its details have remained largely unknown. Therefore, in this study, we immunolocalized therapeutic factors and evaluated the data to study the clinical significance of the molecules in non-functioning Japanese gastrointestinal NET. Fifty-two NET cases were available for examination in this study and expression of somatostatin receptor (sstr) 1, 2A, 2B, 3 and 5, activated form of mammalian target of rapamycin (mTOR), eukaryotic initiation factor 4-binding protein 1 (4EBP1), ribosomal protein s6 (S6), extracellular signal-regulated kinase (ERK) and insulin-like growth factor 1 receptor (IGF-1R) was evaluated using immunohistochemistry. We then studied the correlation among the immunohistochemical results of the individual cases using hierarchical clustering analysis. Results of clustering analysis demonstrated that NET cases were basically classified into Cluster I and II. Cluster I was associated with higher expression of sstr1, 2B and 3 and Cluster II was characterized by an activation of the PI3K/Akt pathway and IGF-1R and higher proliferative status. Cluster II was further classified into Cluster IIa and IIb. Cluster IIa was associated with higher expression of sstr1 and 5 and higher proliferative status and Cluster IIb was characterized by ERK activation. Hierarchical clustering analysis of immunoreactivity of the therapeutic factors can classify NET cases into three distinctive groups and the medical treatment may be determined according to this novel classification method for non-functioning NET patients.

Changes in microRNA expression levels correlate with clinicopathological features and prognoses in endometrial serous adenocarcinomas.

This study aimed to determine the expression profiles of microRNAs (miRNAs) in endometrial serous adenocarcinoma and to examine the association between miRNA expression and clinical outcomes. Twenty-one patients diagnosed with endometrial serous adenocarcinoma between January 2001 and December 2006 were enrolled. miRNA expression profiles were examined using miRNA microarray and qRT-PCR. miRNA expression levels were correlated with clinicopathological variables and survival rates. A total of 120 miRNAs were differentially expressed in endometrial serous adenocarcinoma compared to normal endometria. Of these, 54 miRNAs were down-regulated (>2-fold), including miR-101, miR-10b*, miR-152, and miR-29b, and the remainder were up-regulated (>2-fold), including miR-200a, miR-200b, and miR-205. Decreased expression of miR-10b*, miR-29b, and miR-455-5p was correlated with vascular invasion (P = 0.048, P = 0.013, and P = 0.032, respectively). Univariate analysis revealed that lower expression of miR-101, miR-10b*, miR-139-5p, miR-152, miR-29b, and miR-455-5p was significantly correlated with poor overall survival (P < 0.05), and reduced expression of miR-152, miR-29b, and miR-455-5p was significantly correlated with poor disease-free survival (P < 0.05). Multivariate analysis demonstrated that decreased expression of miR-152 (P = 0.021) was a statistically independent risk factor for overall survival, and decreased expression levels of miR-101 (P = 0.016) and miR-152 (P = 0.010) were statistically independent risk factors for disease-free survival. In addition, transfection of miR-101 or miR-152 precursors into an endometrial serous carcinoma cell line inhibited cell growth (P < 0.0001 and P = 0.01, respectively). Moreover, strong positive immunoreactivity of cyclooxygenase-2 (COX-2) was significantly correlated with down-regulation of miR-101 (P = 0.035). These findings suggest that the dysregulation of miRNAs is associated with the poor prognosis in endometrial serous adenocarcinoma patients.

Steroid and xenobiotic receptor in human esophageal squamous cell carcinoma: a potent prognostic factor.

Steroid and xenobiotic receptor (SXR) is a nuclear receptor activated by diverse exogenous and endogenous compounds and has been demonstrated to play a pivotal role in detoxification through its regulation of various metabolizing enzymes and transporters. Recent studies also demonstrated the potential roles of SXR in the regulation of apoptosis and inflammation in various carcinoma cells, but the status of SXR in human esophageal squamous cell carcinoma (ESCC) has not been examined. Therefore, in this study, we performed immunohistochemical and quantitative RT-PCR evaluations in human ESCC in order to clarify its biological and clinical significance. We first immunolocalized SXR in 73 human ESCC cases. SXR immunoreactivity was detected in the nuclei, or in both nuclei and cytoplasm of carcinoma cells (98%, 20% of cases, respectively). The status of nuclear SXR immunoreactivity was inversely correlated with histological grade, lymph node status, ki67/MIB1 labeling index, and positively correlated with retinoid X receptor alpha status. In addition, high nuclear SXR expression was significantly correlated with favorable clinical outcome of the patients. Multivariate analysis further demonstrated SXR status in carcinoma cells as an independent favorable prognostic factor of the patients. Results of quantitative RT-PCR study demonstrated that SXR mRNA expression was detected in three of five cases, and was marked higher in the cancerous tissue than non-neoplastic tissue of these patients. This is the first study to demonstrate the status of SXR in human ESCC and the results suggest that SXR is a potent favorable prognostic factor of human ESCC.

Immunolocalization of estrogen-producing and metabolizing enzymes in benign breast disease: comparison with normal breast and breast carcinoma.

It is well known that estrogens play important roles in the cell proliferation of breast carcinoma. Benign breast disease (BBD) contains a wide spectrum of diseases, and some are considered an important risk factor for subsequent breast carcinoma development. However, the significance of estrogens in BBD has remained largely unknown. Therefore, in this study, we examined tissue concentrations of estrogens and immunolocalization of estrogen-producing/metabolizing enzymes in BBD, and compared these findings with those in the normal breast and ductal carcinoma in situ (DCIS). Tissue concentration of estradiol in BBD (n = 9) was significantly (3.4-fold) higher than normal breast (n = 9) and nearly the same (0.7-fold) as in DCIS (n = 9). Immunoreactivity of estrogen sulfotransferase in BBD was significantly lower (n = 82) than normal breast (n = 28) but was not significantly different from DCIS (n = 28). Aromatase and steroid sulfatase immunoreactivities tended to be higher (P = 0.07) in BBD than in normal breast, and 17ß-hydroxysteroid dehydrogenase type 1 immunoreactivity was significantly higher in BBD than normal breast in the postmenopausal tissues. Immunoreactivity of estrogen and progesterone receptors was also significantly higher in BBD than normal breast. These results suggest that tissue concentration of estradiol is increased in BBD at a level similar to DCIS, which is considered mainly due to loss of estrogen sulfotransferase expression. Increased local estradiol concentration in BBD due to aberrant expression of estrogen-producing/metabolizing enzymes may play important roles in the accumulation of estradiol-mediated growth and/or subsequent development of breast carcinoma.

Runx2 in human breast carcinoma: its potential roles in cancer progression.

Runx2 has been proposed as one of the pivotal factors in the process of osteogenesis and metastasis in human malignancies including breast cancer, but its details have not been evaluated. Therefore, in this study, we evaluated its expression in human breast cancer using immunohistochemistry. One hundred and thirty-seven formalin-fixed and paraffin-embedded breast cancer specimens were used in this analysis of immunohistochemical study. Immunoreactivity was evaluated using the labeling index (LI). Runx2 immunoreactivity was detected in both carcinoma and stromal cells, as well as non-pathological ductal cells. The nuclear LI of Runx2 in carcinoma cells was associated with the clinical stage, histological grade and HER2 status of the patients examined. In addition, among the patients not associated with distant metastasis, those with high Runx2 LI demonstrated a significantly worse clinical outcome than those with a low LI. This was more pronounced in the group of estrogen receptor (ER)-negative cases. In addition, both univariate and multivariate analyses demonstrated that the Runx2 LI in breast carcinoma cells turned out an independent prognostic factor. Results of our present study demonstrated that Runx2 plays very important roles in the progression of breast cancer, especially in those of ER-negative cases.

Steroid and Xenobiotic Receptor (SXR) as a possible prognostic marker in epithelial ovarian cancer.

We examined the expression of the steroid and xenobiotic receptor (SXR) and evaluated its clinical significance in human epithelial ovarian carcinoma. One hundred forty-one cases were examined using immunohistochemistry for SXR with archival specimens. All cases were scored using a semi-quantitative histological scoring (HSCORE) method. Specimens with an HSCORE > 60 were regarded as SXR-positive. Various clinicopathologic variables were examined. SXR showed significant differences in age, histology, grade, ER alpha and PR. SXR was detected in 35 of 141 (24.8%) ovarian cancer tissues. There was a statistically significant negative correlation between SXR-positive status and both disease-free survival and overall survival (P= 0.0415 and 0.0316, respectively), independent of stage (P= 0.0167 and 0.021, respectively). In multivariate analysis, SXR was a statistically independent risk factor for both disease-free survival and overall survival (P= 0.049 and 0.0354). Our results support an association of SXR between ER alpha and PR in epithelial ovarian cancers. Our data suggest that SXR is a prognostic factor in epithelial ovarian cancer and may represent a useful marker to identify patients at risk of recurrence or death.

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 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.

An analysis of potential surrogate markers of target-specific therapy in archival materials of adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is a rare neoplasm but some of the cases are highly malignant. Clinical outcome of the patients with advanced ACC still remained poor or dismal despite recent development of aggressive antitumor therapies. Target-specific therapies have been developed in a number of human malignancies and resulted in therapeutic benefits in some cancer patients. However, these therapies are only effective in the cases in which corresponding targets are expressed in tumor tissues. Therefore, we evaluated expression of potential surrogate markers using immunohistochemistry in archival materials of adrenocortical carcinoma in order to explore the potential application of target specific therapies in ACC in this study. We immunolocalized ten established or potential surrogate markers of target-specific therapies, located in the Ras/extracellular signal-regulated kinase and phosphatidylinositol-3 kinase/Akt pathways, in 41 ACC cases, 54 adrenocortical adenoma (ACA) cases, and five nonpathological adrenal glands and correlated the findings with clinicopathological factors of the patients. Among these markers examined, only epidermal growth factor receptor (EGFR) was significantly more abundant in ACC than in ACA (P < 0.01). These findings suggest that the agents which specifically inhibit signal transductions through EGFR such as monoclonal antibodies against EGFR are considered to be worthwhile to be attempted in future clinical studies.

Secondary aortoenteric fistula possibly associated with continuous physical stimulation: a case report and review of the literature.

BACKGROUND: Secondary aortoenteric fistula is a rare but fatal complication after reconstructive surgery for an aortic aneurysm characterized by abdominal pain, fever, hematochezia, and hematemesis, and the mortality rate is high. It has been suggested that it arises due to either continuous physical stimulation or prosthesis infection during primary surgery. We describe an aortoenteric fistula following reconstructive surgery for an abdominal aortic aneurysm together with postmortem pathological findings. CASE PRESENTATION: A 59-year-old Japanese man who had undergone reconstructive surgery for an abdominal aortic aneurysm 20 months earlier presented with the chief complaint of hematochezia and malaise. Esophagogastroduodenoscopy and total colonoscopy revealed only colon diverticula with no bleeding. Contrast-enhanced computed tomography revealed gas within the aneurysm sac and adhesion between the replaced aortic graft and intestinal tract, suggesting a graft infection. After 18 days of antibiotic treatment, he suddenly went into a state of shock, with massive fresh bloody stool and hematemesis, followed by cardiac arrest. An autopsy revealed communication between the artery and the ileum through an ulcerative fistula at the suture line between the left aortic graft branch and the left common iliac artery. Pathological analysis revealed tight adherence between the arterial and intestinal walls, but no marked sign of infection around the fistula, suggesting that the fistula had arisen due to physical stimuli. CONCLUSIONS: Pathological analysis suggested that the present secondary aortoenteric fistula arose due to physical stimuli. This reaffirms the importance of keeping reconstructed aortas isolated from the intestine after abdominal aortic aneurysm surgery.

Intratumoral concentration of sex steroids and expression of sex steroid-producing enzymes in ductal carcinoma in situ of human breast.

It is well known that sex steroids play important roles in the development of invasive ductal carcinoma (IDC) of the human breast. However, biological significance of sex steroids remains largely unclear in ductal carcinoma in situ (DCIS), regarded as a precursor lesion of IDC, which is partly due to the fact that the intratumoral concentration of sex steroids has not been examined in DCIS. Therefore, in this study, we first examined the intratumoral concentrations of estradiol and 5alpha-dihydrotestosterone (DHT) using liquid chromatography/electrospray tandem mass spectrometry in DCIS. Intratumoral concentrations of both estradiol and DHT were threefold higher in DCIS than non-neoplastic breast tissues and estrogen-producing enzymes (aromatase, steroid sulfatase, and 17beta-hydroxysteroid dehydrogenase type 1 (17betaHSD1)), and androgen-producing enzymes (17betaHSD5 and 5alpha-reductase type 1 (5alphaRed1)) were abundantly expressed in DCIS by real-time PCR and immunohistochemical analyses. The intratumoral concentration of DHT was significantly lower in IDC than DCIS, while the expression of aromatase mRNA in carcinoma cells and intratumoral stromal cells was significantly higher in IDC than those in DCIS. Immunohistochemistry for sex steroid-producing enzymes in DCIS demonstrated that 5alphaRed1 immunoreactivity was positively correlated with Ki-67 labeling index and histological grade and was also associated with an increased risk of recurrence in patients with DCIS examined. Results of our study suggest that intratumoral concentrations of estradiol and DHT are increased in DCIS, which is possibly due to intratumoral production of these steroids. Therefore, estradiol and DHT may play important roles in the development of DCIS of the human breast.

Loss of estrogen receptor beta isoform expression and its correlation with aberrant DNA methylation of the 5'-untranslated region in human epithelial ovarian carcinoma.

Evidence exists that sex steroids such as estrogens affect epithelial ovarian cancer. The expression profiles of the estrogen receptors (ER) and ERbeta in particular have not been fully described. Therefore, in our present study, we examined the methylation status of the promoters 0K and 0N, and the expression of ERbeta isoforms in human epithelial ovarian carcinoma. We then correlated methylation status with ER expression status. Twelve ovarian carcinoma cell lines, six primary cultures of ovarian surface epithelial cells (OSE), and 64 cases of ovarian carcinoma tissues were examined. Bisulfite sequencing and quantitative reverse transcription-polymerase chain reaction were used to evaluate methylation status and expression of ERbeta isoforms. The relative abundance of exon 0N, ERbeta1, ERbeta2, and ERbeta4 mRNA was significantly lower in ovarian cancer cell lines and tissues than in their corresponding normal counterparts. However, ERbeta5 mRNA level was relatively higher in the cancers, in clear cell adenocarcinoma in particular, than in the normal ovary. Bisulfite sequencing analysis demonstrated that the two promoters of the ERbeta gene exhibited distinct methylation patterns. Promoter 0N was unmethylated in OSE, rarely methylated in normal ovarian tissues, and extensively methylated in ovarian cancer cell lines and tissues (11/15 cell lines and 18/32 cancer tissues were extensively methylated). The promoter 0K was, however, unmethylated in both normal and malignant ovarian cells and tissues. A significant correlation between promoter 0N hypermethylation and the loss of exon 0N, ERbeta1, ERbeta2, and ERbeta4 mRNA expression was detected in ovarian carcinoma cells and tissues. Treatment of ovarian carcinoma cells with 5-aza-2' deoxycytidine resulted in reexpression of the ERbeta gene. The results of our present study suggest that ERbeta is inactivated mainly through aberrant DNA methylation. This process may play an important role in the pathogenesis of epithelial ovarian cancer.

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-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.