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Aims It has been reported that the expression of core 2 β1,6-N-acetylglucosaminyl transferase 1 (C2GnT1), which synthesizes the core 2 branching structure on O-glycans, may be associated with the biological aggressiveness of tumour cells. Therefore, the aim of this study was to examine the relationship between the expression of C2GnT1 and clinicopathological parameters of patients with endometrial carcinoma. Methods and results The immunohistochemical expression of C2GnT1 was examined in 84 cases of endometrioid-type endometrial carcinoma, 15 cases of endometrial hyperplasia, and 30 normal endometria. The staining intensity was reported according to a positivity index (PI, full score 100), calculated from the percentage of positive cells. The expression of C2GnT1 was significantly higher in endometrial carcinoma (PI = 8.31 ± 15.29) than in normal endometrium (PI = 0.52 ± 1.24) (P

Placental mesenchymal dysplasia (PMD) is characterized by multiple hypoechoic vesicles which are similar to molar changes in the placenta; however, the process of such morphological changes of PMD during pregnancy has not been fully understood. We performed a review of all PMD cases published in English and identified 49 articles including 110 cases. With regard to the gestational age at which the multicystic pattern was seen, approximately 70% of cases were diagnosed at 13-20 weeks of gestation. Another characteristic feature of PMD is varicose dilation of fetal chorionic vessels. As many as 90% of cases were diagnosed as placenta with dilated fetal chorionic vessels in the third trimester. We also report a case of PMD which was found at 10 weeks of gestation according to ultrasonic molar patterns. Serial observations of the placenta using ultrasound and magnetic resonance imaging revealed that multicystic lesions became smaller after 23 weeks. In contrast, dilated placental vessels on the fetal side became apparent at 38 weeks. The present review highlights that placental vesicular lesions of PMD may precede dilation of fetal chorionic vessels during pregnancy. It also indicates the potential of a gradual reduction in size of PMD's placental vesicular lesions by serial study of placental images., Article, GYNECOLOGIC AND OBSTETRIC INVESTIGATION. 75(4):217-223 (2013)

Although progestin has been used to treat endometrial hyperplasia and endometrial carcinoma (EC), its therapeutic efficacy is limited. In order to improve this, the underlining mechanisms of the effects of progestin need to be elucidated in more detail. In the present study, we examined the involvement of mitogen-inducible gene-6 (MIG6), a negative regulator of the EGF receptor, in the progestin-mediated growth suppression of endometrial epithelia. The immunohistochemical expression of MIG6 was elevated in the early to mid-secretory phases of normal endometrium and also with endometrial hyperplasia after medroxyprogesterone acetate (MPA) therapy. The addition of progesterone (P4) to progesterone receptor (PR)-positive EC cells reduced the viability and induced MIG6 messenger RNA (mRNA) and protein expression. The silencing of MIG6 using siRNA eliminated the P4-mediated reduction of EC cell viability, indicating that MIG6 is an essential downstream component of PR-mediated growth suppression. In order to enhance PR-driven signals, we examined the effects of histone deacetylase (HDAC) inhibitors because histone acetylation has been shown to increase the expression of PR. The addition of three HDAC inhibitors (panobinostat, LBH589; trichostatin A, TSA; suberoylanilide hydroxamic acid, SAHA) decreased the viability of EC cells and up-regulated the expression of PR and MIG6, and these effects were the strongest with LBH589. The addition of LBH589 and MPA synergistically decreased the viability and increased apoptosis in EC cells. These results indicate that LBH589 has potential as an enhancer of progestin therapy via the up-regulation of PR and MIG6.

Patients with uterine leiomyosarcoma (LMS) typically present with vaginal bleeding, pain, and a pelvic mass, with atypical presentations of hypercalcemia and eosinophilia also being reported. Radiographic evaluation with combined positron-emission tomography/computed tomography may assist in diagnosis and surveillance in women with uterine LMS; these are commonly used with stage and tumour grade as prognostic indicators and a recently developed risk-assessment index to predict disease-specific survival. Recent studies have shown that the addition of adjuvant therapy after surgical management does not seem to improve survival, and ovarian preservation does not appear to negatively impact outcome. Experimentally, it is noteworthy that proteasome subunit beta 9 (PSMB9)/β1i-deficient mice exhibit spontaneous development of uterine LMS, with a disease prevalence of ~37% by 12 months of age. Furthermore, a recent report showed the loss of ability to induce PSMB9/β1i expression, that is up-regulated by interferon-γ (IFNγ), in human uterine LMS tissues. Here, we reviewed human uterine LMS for genetic mutations in the IFNγ signal cascade, and found serious mutations in three genes, Janus activated kinase 1 (JAK1), signal transducer and activator of transcription 1 (STAT1) and PSMB9/β1i promoter regions. Moreover, molecular experiments demonstrated differential expression of cyclin E and P27/KIP1, that regulate cell-cycle G1 arrest via PSMB9/β1i expression. The discovery of this mutational activation of a key cell-signalling pathway may provide new targets for diagnostic approaches and therapeutic intervention.

Takuma Hayashi (Shinshu University School of Medicine) discussed a novel biomarker for detecting human uterine leiomyosarcoma (LMS).

Ovarian clear cell carcinoma (CCC) arises from ovarian endometriosis. Intra-cystic fluid contains abundant amounts of free iron, which causes persistent oxidative stress, a factor that has been suggested to induce malignant transformation. However, the mechanisms linking oxidative stress and carcinogenesis in CCC currently remain unclear. Lipocalin 2 (LCN2), a multifunctional secretory protein, functions as an iron transporter as well as an antioxidant. Therefore, we herein examined the roles of LCN2 in the regulation of intracellular iron concentrations, oxidative stress, DNA damage, and antioxidative functions using LCN2-overexpressing (ES2), and LCN2-silenced (RMG-1) CCC cell lines. The results of calcein staining indicated that the up-regulated expression of LCN2 correlated with increases in intracellular iron concentrations. However, a DCFH-DA assay and 8OHdG staining revealed that LCN2 reduced intracellular levels of reactive oxygen species and DNA damage. Furthermore, the expression of LCN2 suppressed hydrogen peroxide-induced apoptosis and prolonged cell survival, suggesting an antioxidative role for LCN2. The expression of mRNAs and proteins for various oxidative stress-catalyzing enzymes, such as heme oxygenase (HO), superoxide dismutase (SOD), and glutathione peroxidase, was not affected by LCN2, whereas the intracellular concentration of the potent antioxidant, glutathione (GSH), was increased by LCN2. Furthermore, the expression of xCT, a cystine transporter protein, and CD44 variant 8-10 (CD44v), a stem cell marker, was up-regulated by LCN2. Although LCN2 increased intracellular iron concentrations, LCN2-induced GSH may catalyze and override oxidative stress via CD44v and xCT, and subsequently enhance the survival of CCC cells in oxidative stress-rich endometriosis.

Purpose Lipocalin 2 (LCN2) is a secretory protein that is involved in various physiological processes including iron transport. We previously identified LCN2 as an up-regulated gene in endometrial carcinoma, and found that the overexpression of LCN2 and its receptor, SLC22A17, was associated with a poor prognosis. However, the functions and mechanism of action of LCN2 currently remain unclear. Methods The LCN2-overexpressing endometrial carcinoma cell lines, HHUA and RL95-2, and LCN2-low-expressing one, HEC1B, were used. The effects of LCN2 on cell migration, cell viability, and apoptosis under various stresses, including ultraviolet (UV) irradiation and cisplatin treatment, were examined using the scratch wound healing assay, WST-1 assay, and Apostrand assay, respectively. Results LCN2-silencing using shRNA method significantly reduced the migration ability of cells (p

Inflammation in the ovary, including ovulation and pelvic inflammatory disease, has been proposed to play a role in the pathogenesis of ovarian cancer. Endometriotic lesions trigger a local inflammatory reaction and have been reported to be associated with an increased risk of epithelial ovarian cancer. However, the precise molecular mechanisms of ovarian cancer arising from endometriosis are still to be elucidated. To clarify the involvement of mismatch repair (MMR) abnormalities in the inflammation-associated malignant transformation of endometriosis, the immunohistochemical expression of mismatch repair proteins (human mutL homolog 1 [hMLH1] and human mutS homolog 2 [hMSH2]) was examined in 27 cases of ovarian endometriosis, 25 cases of ovarian carcinoma accompanied by endometriosis, and 39 cases of solitary ovarian carcinoma. In addition, the relationship between mismatch repair abnormalities including the microsatellite instability, PTEN (phosphatase and tensin homolog) mutation, and clinicopathologic parameters was analyzed. The expression of mismatch repair proteins was stepwisely decreased in endometriosis, ovarian carcinoma accompanied by endometriosis, and ovarian carcinoma. Tumors harboring multiple microsatellite instability (high-frequency microsatellite instability [MSI-H]) were detected in 4 (14.8%) of 27 cases of endometriosis and 7 (30.4%) of 23 cases of ovarian carcinomas. The frequency of PTEN mutations was higher in MSI-H cases than in microsatellite instability-stable (MSI-S) cases. In 2 cases of ovarian carcinoma accompanied by endometriosis, the decreased expression of mismatch repair proteins and MSI-H was observed in both the endometriosis and carcinoma lesions. Clinicopathologically, the MSI-H cases were associated with elevated serum levels of C-reactive protein and higher white blood cell counts. These findings suggest that mismatch repair abnormalities might be involved in the malignant transformation of ovarian endometriosis and that inflammation induces mismatch repair abnormalities during ovarian carcinogenesis arising from endometriosis.

Uterine leiomyosarcoma (Ut-LMS) develops more often in the muscle tissue layer of the uterine body than in the uterine cervix. The development of gynecologic tumors is often correlated with female hormone secretion; however, the development of Ut-LMS is not substantially correlated with hormonal conditions, and the risk factors are not yet known. Importantly, a diagnostic-biomarker which distinguishes malignant Ut-LMS from other uterine mesenchymal tumors including leiomyoma (LMA) is yet to be established. Accordingly, it is necessary to analyze risk factors associated with Ut-LMS, to establish a clinical treatment method. Proteasome subunit, low-molecular mass polypeptide(LMP2)/b1i-deficient mice spontaneously develop Ut-LMS, with a disease prevalence of ~40% by 14 months of age. Recent experiments with human and mouse uterine tissues revealed defective LMP2/b1i expression in human Ut-LMS that was traced to the interferon (IFN)-g pathway and a specific effect of Janus kinase (JAK)-1 somatic mutations on LMP2/b1i transcriptional activation. Furthermore, analysis of a human Ut-LMS cell line clarified the biological significance of LMP2/b1i in malignant myometrium transformation and the cell cycle, thus implicating LMP2/b1i as an anti-tumorigenic candidate. Therefore, defective-LMP2/b1i expression may be a risk factor for human Ut-LMS. LMP2/b1i is a potential diagnostic-biomarker for Ut-LMS, and may be a targeted-molecule for a new clinical therapeutic approach.

Uterine leiomyosarcoma (LMS) is a highly metastatic smooth muscle neoplasm for which calponin h1 is suspected to have a biological role as a tumor-suppressor. We earlier reported that LMP2-null mice spontaneously develop uterine LMS through malignant transformation of the myometrium, thus implicating this protein as an anti-tumorigenic candidate as well. In the present study, we show that LMP2 may negatively regulate LMS independently of its role in the proteasome. Moreover, several lines of evidence indicate that although calponin h1 does not directly influence tumorigenesis, it clearly affects LMP2-induced cellular morphological changes. Modulation of LMP2 may lead to new therapeutic approaches in human uterine LMS., Article, FEBS LETTERS. 586(13):1824-1831 (2012)

Hypoxia is known to play important roles in the development and progression of tumors. We previously demonstrated that S100A4, a critical molecule for metastasis, was upregulated in ovarian cancer cells. Therefore, we examined the mechanisms of the upregulation of S100A4 expression in ovarian carcinoma cells, with particular attention paid to the effects of hypoxia. The expression levels of S100A4 were found to be correlated with the invasiveness of ovarian carcinoma cells in vitro and in vivo, and the upregulation of S100A4 expression was associated with hypomethylation of CpG sites in the first intron of S100A4 in ovarian carcinoma cell lines and tissues. The expression of S100A4 was increased under hypoxia and was associated with elevated invasiveness, which was inhibited by S100A4 small interfering RNA (siRNA). In addition, exposure to hypoxia reduced the methylation of hypoxia-response elements (HRE) of the S100A4 gene in a time-dependent fashion, in association with the increased binding of HIF-1α to a methylation-free HRE in ovarian carcinoma cells. These results indicate that hypoxia-induced hypomethylation plays an essential role in S100A4 overexpression and the epigenetic transformation of ovarian carcinoma cells into the "metastatic phenotype."

Mitsuhashi Y, Horiuchi A, Miyamoto T, Kashima H, Suzuki A & Shiozawa T (2012) Histopathology 60, 826–837 Prognostic significance of Notch signalling molecules and their involvement in the invasiveness of endometrial carcinoma cells Aims: The aim of this study was to investigate the significance of the expression of Notch-related molecules in endometrial carcinoma. Methods and results: The expression of Notch receptors (Notch1 and 3) and Notch ligands [Jagged (JAG) 1 and Delta-like (DLL) 4] was examined immunohistochemically in 37 normal and 76 malignant endometrial tissue samples. For each section, immunohistochemical staining was scored using a positivity index (PI, full score; 200). The effects of a Notch inhibitor, DAPT, on cell proliferation, invasion and motility were investigated using endometrial carcinoma cell lines. The PIs for Notch1 (mean ± SD 90.4 ± 15.3), Notch3 (95.6 ± 20.4), JAG1 (95.5 ± 10.0) and DLL4 (88.2 ± 9.6), were significantly higher in endometrial carcinoma than normal endometrium. The PI for Notch1 was associated significantly with advanced International Federation of Gynecologists & Obstetricians (FIGO) stage. In addition, patients with tumours showing high expression of both Notch1 and JAG1 had a poor prognosis compared with those having double-negative carcinomas (P = 0.015). DAPT suppressed invasiveness of cells derived from the endometrial carcinoma cell line KLE. Conclusions: The Notch1–JAG1 axis may enhance the invasive properties of endometrial carcinomas, which suggests the Notch pathway may be a promising target for the treatment of this malignancy.

Background: Although the majority of smooth muscle neoplasms found in the uterus are benign, uterine leiomyosarcoma is extremely malignant, with high rates of recurrence and metastasis. The development of gynecologic tumors is often correlated with secretion of female hormone; however, the development of human uterine leiomyosarcoma is not substantially correlated with hormonal conditions, and the risk factors are unclearly understood. Importantly, a diagnostic-biomarker, which distinguishes malignant human uterine leiomyosarcoma from benign tumor leiomyoma is yet to be established. Aims : It is necessary to analyze risk factors associated with human uterine leiomyosarcoma, in order to establish a diagnostic-biomarker and a clinical treatment method. Patients and Methods : Histology and Immunofluorescence Staining: Uteri obtained from LMP2 -/- mice or its parental mice (C57BL/6 mice) were fixed in 10% buffered formalin, incubated in 4% paraformaldehyde for 8 hours, and embedded in paraffin. Tissue sections (5 μm) were prepared and stained with H&E for routine histological examination or were processed further for immunofluorescence staining with appropriate antidodies. Furthermore, a total of 101 patients between 32 and 83 years of age and diagnosed as having smooth muscle tumors of the uterus were selected from pathological files. Immunohistochemistry staining for LMP2 was performed on serial human uterine leiomyosarcoma, leiomyoma and myometrium sections. Results : Homozygous deficient mice for a proteasome β1i subunit, LMP2 spontaneously develop uterine leiomyosarcoma, with a disease prevalence of ~40% by 14 months of age. Defective LMP2 expression in human uterine leiomyosarcoma was demonstrated, but present in human leiomyoma and myometrium. Conclusions : Loss in LMP2 expression may be one of the risk factors for human uterine leiomyosarcoma. LMP2 may be a potential diagnostic-biomarker and targeted-molecule for a new therapeutic approach.

Sarcomas are a rare form of malignant tumor, with less than 15,000 new cases being diagnosed each year in the United States. In spite of their rarity, sarcomas are highly debilitating malignancies that are often associated with significant morbidity and mortality. They are also biologically very heterogeneous because they originate from many different tissues and cell types. Sarcomas have classically been defined by their tissue of origin and are additionally stratified according to their histopathology or the age of the patient at diagnosis. Uterine mesenchymal tumors that develop in the myometrium have traditionally been divided into benign uterine usual LMA, cellular LMA and malignant Ut-LMS based on cytological atypia, mitotic activity and other criteria. Ut-LMS is relatively rare, having an estimated annual incidence of 0.64 per 100,000 women [1], and is resistant to chemotherapy and radiotherapy; therefore, surgical interventions are virtually the only means of treatment [2, 3]. The prognosis of patients with Ut-LMS is poor, and the 5-year survival rate is approximately 35%. Uterine LMA may occur in 70-80% of women by the age of 50 years [4]. Difficulties have been reported in distinguishing Ut-LMS from other uterine mesenchymal tumors including uterine LMA, and a diagnosis generally requires surgery and cytoscopy. Diagnostic categories for uterine mesenchymal tumors and morphological criteria are used to assign cases. The non-standard subtypes of uterine mesenchymal tumors such as the epithelioid and myxoid types are classified in a different manner using these features; therefore, a diagnostic method needs to be established that can identify non-standard smooth muscle differentiation [5, 6]. High estrogen levels have been shown to significantly influence the development of tumors in the uterine body [7]. However, the molecular mechanisms underlying the transformation of uterine LMA and development of Ut-LMS remain unknown. Tumors that have developed and grown in the myometrium increase in size due to the influence of the female hormone, estrogen, which leads to the generation of more tumors. However, a relationship has not yet been reported between the development of Ut-LMS and hormonal conditions, and no obvious risk factors have been identified. The identification of risk factors associated with the development of human Ut-LMS will contribute significantly to the development of preventive and therapeutic treatments. Cytoplasmic proteins are mostly degraded by a protease complex referred to as the 20S proteasome, which has many substrates that consist of twenty-eight 20 to 30 kDa subunits [8, 9]. Proteasomal degradation is essential for many cellular processes, including the cell cycle, regulation of gene expression and immunological function [10]. A previous study reported that an interferon (IFN)-γ treatment induced the expression of large numbers of responsive genes, the β-ring subunits of proteasomes, i.e., low-molecular mass polypeptide (LMP)2/β1i, LMP7/β5i and LMP10/multicatalytic endopeptidase complex-like (MECL)-1/β2i [11]. Ut-LMS was detected in female LMP2/β1ideficient mice at 6 months or older, and its incidence at 14 months was approximately 40% [12]. Histopathological studies of LMP2/ β1i-lacking uterine tumors have revealed the characteristic abnormalities of Ut-LMS [12]. The non-standard subtypes of uterine mesenchymal tumors such as the epithelioid and myxoid types have been classified in a Manuscript accepted for publication May 15, 2014

Immature teratoma of the ovary is an uncommon tumor comprising 1% of ovarian malignancies. The amount of immature neuroepithelium in the teratoma is an important prognostic factor. Although the current grading system based on this criterion is widely accepted, the biological significance of immature neuroepithelium is poorly understood. In this study, we used immunohistochemistry to evaluate the expression of Oct4 (also known as Oct3 or POU5F1), a transcription factor expressed in primordial germ cells and embryonic stem cells, along with that of PAX6, a transcription factor contributing to neurogenesis, and CD56, a known marker for tumors of neural origin, in 18 cases of pure immature teratoma of the ovary. Oct4 was expressed in the immature neuroepithelium of all 7 grade-3 cases and 2 grade-2 cases. It was not expressed in 4 grade-2 cases and all 5 grade-1 cases. These tumor cells lacked CD30 or α-fetoprotein expression, which supported the diagnosis of pure immature teratoma. PAX6 was expressed in the immature neuroepithelium of all immature teratomas, but not in Oct4-positive cells. CD56 was expressed in neural components of various maturities including PAX6-positive immature neuroepithelium, but not in Oct4-positive cells. These data suggest that the expression of these markers probably reflects the differentiation status of neural tissue in immature teratomas. The finding that Oct4 expression was exclusively detected in immature neuroepithelium of high-grade immature teratomas indicates that Oct4 might serve as a promising biomarker for the diagnosis of highly malignant cases of immature teratoma.

Uterine tumors are the most common type of gynecologic neoplasm. Uterine leiomyosarcoma (LMS) is rare, accounting for 2% to 5% of tumors of the uterine body. Uterine LMS develops more often in the muscle tissue layer of the uterine body than in the uterine cervix. The development of gynecologic tumors is often correlated with female hormone secretion; however, the development of uterine LMS is not substantially correlated with hormonal conditions, and the risk factors are not yet known. Radiographic evaluation combined with PET/CT can be useless in the diagnosis and surveillance of uterine LMS. Importantly, a diagnostic biomarker, which distinguishes malignant LMS and benign tumor leiomyoma (LMA) is yet to be established. Accordingly, it is necessary to analyze risk factors associated with uterine LMS in order to establish a method of treatment. LMP2-deficient mice spontaneously develop uterine LMS, with a disease prevalence of ∼40% by 14 months of age. It is therefore of interest whether human uterine LMS shows a loss of LMP2 expression. We found LMP2 expression is absent in human LMS, but present in human LMA. Therefore, defective LMP2 expression may be one of the risk factors for LMS. LMP2 is potentially a diagnostic biomarker for uterine LMS, and gene therapy with LMP2-encording DNA may be a new therapeutic approach.

Background: Ovarian clear cell carcinoma (OCCC) is one of the 4 major histological types of ovarian carcinoma (OVC) and is frequently arising from ovarian endometriotic cysts. OCCC is rare in the United States and western countries, but it is more frequent in Japan and may be seen in almost 25% of OVC. OCCC commonly shows chemo-resistance which makes difficult to treat OCCC. In order to develop the new therapeutics for OCCC, genetic abnormalities that lead to the development of OCCC need to be elucidated in detail. The comprehensive analysis using next-generation sequencing has rapidly revealed the accumulation of genetic abnormalities in many cancers. However, the significance of each abnormality has not been elucidated. Therefore, functional screening using a cDNA expression library was performed in order to identify genes involved in the pathogenesis of OCCC. Methods: The mRNA was extracted from an OCCC cell line, RMG-1, and a cDNA library was established using a retroviral vector. This cDNA library was transfected into mouse NIH3T3 cells, and then the transformed foci which formed multilayered growth were picked up and the inserted DNAs of those foci were detected by sequencing. The mRNA and protein expression and the transforming ability of the identified genes were confirmed by immunohistochemistry and focus-formation assay. Result: Seven genes were isolated; Dishevelled 1(DVL1), beta subunit of Sec61 (SEC61B), K-specific demethylase 4A (KDM4A), chromosome 1 open reading frame 38 (C1ORF38), splicing factor 1 (SF1), ribosomal protein S2 (RPS2), and ribosomal protein S3A (RPS3A). Of these genes, RT-PCR and immunostaining revealed that the expression of SEC61B and C1ORF38 was stronger than that in other histological types of ovarian cancer. A mutational analysis of the DVL1 c-terminal region detected 3 point-mutations in RMG1 cells, whereas no mutation was found in 24 cases of OCCC. Focus formation was confirmed by the transfection of SEC61B, C1ORF38, and DVL1 into NIH3T3 cells. Conclusion: These results suggest that SEC61B, C1ORF38, and DVL1 may be involved in the pathogenesis of OCCC. Citation Format: Tsutomu Miyamoto, Yasushi Yamada, Koichi Ida, Ryoichi Asaka, Hisanori Kobara, Hirofumi Ando, Hiroyasu Kashima, Akihisa Suzuki, Tanri Shiozawa. Functional screening of genes involved in carcinogenesis using a cDNA expression library of ovarian clear cell carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3091.

Background: Although estrogen has been regarded as a risk factor for endometrial carcinoma, its concentration-dependent carcinogenetic effects remain unclear, because most endometrial carcinomas occur in post-menopausal women, whose serum estrogen levels are relatively low. Because impaired mismatch repair (MMR) was reportedly crucial in the early stages of endometrial carcinogenesis, we focused on the relationship between various estrogen concentration and MMR activity in normal and malignant endometrial glandular cells. In those cells, we reported that the protein and mRNA expression of both hMLH1 and hMSH2, the key factors of MMR, were up-regulated by estrogen stimulation. In addition, we demonstrated that the estrogen stimulation enhanced the MMR activity in those cells using in vitro MMR assay with the M13mp2 phage heteroduplex DNAs and that the proliferating cells without hMLH1/hMSH2 expressions implying “high risk” cells were more frequently observed under relative-low estrogen concentrations. In this study, to further confirm the role of E2 in vivo, we employed a mouse endometrial carcinoma model induced by intra-uterine injection of a carcinogen, N-methyl-N-nitrosourea (MNU) under various E2 concentrations. Methods: MNU was injected into the uterine cavity of 29 CD-1 mice at 6 weeks of age, and E2 was administered by pellets or orally. Uteri were removed for histological examinations 24 weeks later, and serum E2 levels were measured. The immunohistochemical expression of MMR proteins in uterine epithelia was investigated. Results: Of 29 mice, 8, 8, 8, and 5 showed atrophic, normal, hyperplastic, and carcinomatous endometria, respectively. The mean E2 levels of each group were 0.2pg/ml, 3.8pg/ml, 190.0pg/ml, and 6.7pg/ml, with significant differences. The expression of the MMR proteins was stronger in mice with elevated E2. Conclusions: Elevated E2 levels preferentially induced endometrial hyperplasia rather than carcinoma, and this may be mediated by MMR proteins. These results indicate that modest E2 is needed, whereas elevated E2 levels are not necessarily advantageous for carcinogenesis, suggesting the importance of low-chronic (un-opposed) estrogen in human endometrial carcinogenesis. Citation Format: Ryoichi Asaka, Tsutomu Miyamoto, Koichi Ida, Hodaka Takeuchi, Hirofumi Ando, Yasushi Yamada, David Hamisi Mvunta, Hisanori Kobara, Hiroyasu Kashima, Tanri Shiozawa. Suppressive role of elevated estrogen on endometrial carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5227.

Summary Overexpression of histone deacetylases has been reported in various human malignancies; however, the expression of histone deacetylases in endometrial tissue is not fully understood. In the present study, the expression of histone deacetylase 1, histone deacetylase 2, and Ki-67 was examined immunohistochemically in 30 normal and 66 malignant endometrial tissue samples. The results were expressed as a positivity index and compared with the positivity index for Ki-67 and rates of patient survival. The effect of 2 histone deacetylase inhibitors, trichostatin A and apicidine, on cell proliferation and the expression of cell cycle regulators such as cyclins (D1, E, and A), p21, p27, and p16 were investigated using 6 endometrial carcinoma cell lines. The positivity index for histone deacetylase 1 (79.8 ± 33.0, mean ± SD) and histone deacetylase 2 (106.3 ± 41.9) was higher in endometrial carcinoma than the normal endometrium, with a significant difference for histone deacetylase 2. The positivity index for histone deacetylase 2 was significantly increased in higher-grade carcinomas (positivity index for grade 3, 124.9 ± 28.4) compared with grade 1 tumors (86.0 ± 41.0) and was positively correlated with that for Ki-67. In addition, patients with histone deacetylase 2–positive carcinomas had a poor prognosis compared with those with histone deacetylase 2–negative carcinoma ( P = .048). Treatment with trichostatin A or apicidine suppressed the proliferation in all cell lines examined, in association with increased expression of p21 and down-regulation of cyclin D1 and cyclin A expression. These results indicated that increased histone deacetylase 2 expression is involved in the acquisition of aggressive behavior by endometrial carcinoma and suggest histone deacetylase inhibitor to be a promising anticancer drug for this carcinoma.

Copyright (c) 2010 Taylor & Francis. This is an electronic version of an article published in "GYNECOLOGICAL ENDOCRINOLOGY, Volume 26, Issue 3, pages 220-229 (2010)". GYNECOLOGICAL ENDOCRINOLOGY is available online at: https://doi.org/10.3109/09513590903215482, Cyclin-dependent-kinase (cdk) inhibitor, p27, Article, GYNECOLOGICAL ENDOCRINOLOGY. 26(3):220-229 (2010)

Most fetal goitrous hypothyroidisms are reportedly caused by the maternal use of an antithyroid drug or fetal dyshormonogenesis. However, fetal goitrous hypothyroidism due to the transplacental passage of maternal thyroid stimulation-blocking antibody (TSBAb) is extremely rare. A woman at 28 weeks of gestation was found to have a fetal goiter by ultrasonography. Because the maternal serum showed hypothyroidism with an elevated titer of TSBAb, levothyroxine sodium was administered. The patient delivered a male infant, 3,412 g, with a goiter at term. Umbilical blood revealed primary hypothyroidism with increased TSBAb, and the infant was given levothyroxine sodium. After a month, neonatal thyroid function and TSBAb levels became normal. Attention should be paid to possible fetal hypothyroidism when a fetal goiter is observed to avoid impaired mental development of the neonate., Article, FETAL DIAGNOSIS AND THERAPY. 28(4):220-224 (2010)

Although overexpression of cyclin A2 is reportedly an indicator of a poor prognosis of various malignancies including endometrial carcinoma, its molecular mechanism remains undetermined. To address this issue, we examined the effect of cyclin A2 on the development of resistance to chemotherapeutic drugs. The expression of cyclin A2 protein was increased in advanced-stage and chemotherapy-refractory stage endometrial carcinomas compared with that in early-stage tumours. The expression levels of cyclin A2 in endometrial carcinoma cell lines correlated positively with the IC50 for cisplatin. Endometrial carcinoma HHUA cells that overexpressed cyclin A2 showed increased resistance to cisplatin in vitro and in vivo, via the activation of a survival pathway, the inositol-3 phosphate kinase (PI3K) cascade. The use of a cDNA microarray identified an Akt-binding protein, periplakin, as a novel target of cyclin A2. The cyclin A2-induced up-regulation of periplakin was mediated via direct binding of Sp1 to the promoter that was activated by cyclin A2 along with chromatin remodelling involving CBP/p300, and the siRNA-mediated silencing of periplakin suppressed the PI3K pathway. These results indicate cyclin A2 to be involved in the acquisition of aggressive behaviour of tumour cells through the activation of PI3K by cyclin A2-induced periplakin, and to be a promising therapeutic target.

The hedgehog (Hh) signal pathway has recently been shown to be activated in human malignancies. However, little is known about its role in the development or patient prognosis of epithelial ovarian carcinoma. In the present study, we examined in vivo and in vitro the expression and functional role of Hh signal molecules in epithelial ovarian tumors and normal ovarian surface epithelial (OSE) cells. The expression of Shh, Dhh, Ptch, Smo and Gli1 proteins was not observed in normal OSE, but was increased stepwise in benign, borderline and malignant neoplasms. In addition, immunoreactivity for Shh, Dhh, Ptch, Smo and Gli1 was highly correlated with cell proliferation assessed by Ki-67. Blocking the Hh signal using either the Hh pathway inhibitor cyclopamine or Gli1 siRNA led to remarkably decreased cell proliferation in ovarian carcinoma cells. Treatment with cyclopamine induced not only G, arrest but also apoptosis along with the downregulation of cyclin A and cyclin D1, and the upregulation of p21 and p27. Among the Hh signal molecules, Dhh expression was correlated with poor prognosis of ovarian carcinoma patients. These findings suggest that the Hh signal pathway plays an important role in ovarian tumorigenesis as well as in the activation of cell proliferation in ovarian carcinomas. Thus, the Hh signal pathway is a possible molecular target of new treatment strategies for ovarian carcinoma.

We previously reported the overexpression of cyclins in uterine cervical carcinoma; however, their clinicopathological significance remained undetermined. In the present study, we examined the immunohistochemical expression of cyclins (D1, E, A, B1), p53 and Ki-67 in squamous cell carcinoma (stage Ib+II; 80 cases, stage III+IV; 23 cases). Correlations between the expression of cyclins and clinicopathological parameters and patient survival were statistically evaluated. The results indicated that in the normal squamous epithelium, the expression of cyclins and Ki-67 was sporadically observed in the parabasal layer. Of the 103 cervical carcinomas, overexpression of cyclins D1, E, A, B1 and p53 was observed in 13 (13%), 23 (22%), 25 (24%), 18 (18%) and 23 (22%) cases, respectively, with a slight predominance in advanced stage tumors. The expression of cyclin D1, E, A and p53 significantly correlated with that of Ki-67 (Spearman's rank correlation). Univariate and multivariate analyses revealed that lymph node metastasis and cyclin A overexpression were independent prognostic factors for unfavorable outcomes in stage Ib+II patients. These findings suggest that the overexpression of various cyclins is involved in the acquisition of the vigorous growth potential of cervical carcinoma cells, and that cyclin A is an independent prognosticator of cervical carcinoma in early stages.

E-cadherin and catenins play key roles in cell adhesion and motility. Little is known about the changes in expression of these molecules in the progression of ovarian carcinomas. In the present study, the immunohistochemical expression of E-cadherin and alpha-, beta-, and gamma-catenins was examined in 77 cases of ovarian carcinoma. In addition, the expression of these molecules was evaluated in 26 matched pairs of primary and metastatic lesions of advanced ovarian carcinomas. Of the 77 primary lesions, positive staining for E-cadherin and alpha-, beta-, and gamma-catenin was observed in 75 (97%), 63 (82%), 71 (92%) and 57 (74%) cases, respectively. Positivity for E-cadherin and alpha-, beta-, and gamma-catenin was significantly decreased in stage III and IV tumors compared with stage I and II tumors, suggesting that expression of the cadherin-catenin complex is reduced with the advancing stages of a tumor. Interestingly, expression of E-cadherin and alpha-, beta-, and gamma-catenin in the lesions of peritoneal dissemination was significantly increased compared with the primary lesions. These findings suggest that expression of the cadherin-catenin complex changes markedly and that reexpression may occur during the peritoneal dissemination of ovarian carcinoma cells.

Ovarian tumors associated with hypercalcemia due to ectopic secretion of parathyroid hormone (PTH) are extremely rare. A 33-year-old woman presented with a pelvic mass and profound hypercalcemia accompanied by an elevated serum level of PTH. Laparotomy demonstrated a left ovarian tumor that on histological examination was a neuroendocrine carcinoma of non-small cell type admixed with a component of endometrioid adenocarcinoma. After left salpingo-oophorectomy, the serum calcium and PTH levels normalized. The cells of the neuroendocrine carcinoma were positive for neuron-specific enolase, synaptophysin, chromogranin A, and PTH. Hypercalcemia and elevated serum PTH levels recurred during tumor relapse, and the patient died of disease 6 months postoperatively. This is the eleventh case of neuroendocrine carcinoma of non-small cell type associated with surface epithelial neoplasm of the ovary, and the first such tumor to be associated with hypercalcemia.

To explore the mechanism of estrogen-induced growth of normal endometrium, the transactivation system of the cyclin D1 gene was analysed using cultured normal endometrial glandular cells. Estradiol (E2) treatment of cultured normal endometrial glandular cells induced upregulation of c-Jun, and then cyclin D1 proteins, followed by serial expressions of cyclins E, A and B1 proteins. Increase in the mRNA expression of cyclin D1 preceded the protein expression of cyclin D1 under E2 treatment. A luciferase assay using deletion constructs of the cyclin D1 promoter indicated that E2-induced increase in transcriptional activity was observed in reporters containing AP-1-binding site sequence, and that in the absence of E2, cotransfection of c-Jun also showed increase of transcriptional activity in the same reporters with AP-1 sequence. A gel shift assay using nuclear extract from E2-treated endometrial glandular cells and AP-1 sequences of the cyclin D1 promoter indicated specific binding between c-Jun protein and the promoter. Transfection of c-jun antisense oligonucleotides to the glandular cells resulted in the suppression of the E2-induced upregulation of cyclin D1 mRNA and protein. These findings suggest that E2-induced proliferation of normal endometrial glandular cells is initiated by transcriptional activation of cyclin D1 via binding of c-Jun to the AP-1 sequences.

Background . Endometrial carcinoma associated with pregnancy is rare. We present the case of a 39-year-old woman with endometrial carcinoma in the septate uterus detected 6 months after normal delivery. Case . The patient complained of vaginal bleeding from 32 weeks' gestation, and had a spontaneous vaginal delivery at 37 weeks. The bleeding continued after the delivery, but repeated endometrial cytology and biopsy did not reveal evidence of malignancy. An exploratory laparotomy was performed 6 months postpartum with suspicion of uterine myoma. A histological diagnosis of endometrial carcinoma was made based on examination of frozen sections from the hysterectomy specimen, and bilateral salpingo-oophorectomy and pelvic/para-aortic lymphadenectomy were also performed. Histological examination revealed G3 endometrioid adenocarcinoma with squamous differentiation, which arose in the septum of the uterus and deeply invaded the myometrium. The patient received postoperative chemotherapy and is healthy with no evidence of disease 3 years after the treatment. Conclusion . Although pregnancy-associated endometrial carcinoma is rare, careful examinations are needed when unexplained vaginal bleeding continued.

Minichromosome maintenance (MCM) proteins are essential for cell cycling due to their function as replication-licensing factors. The aim of the present study was to investigate the clinicopathologic implications of the MCM2 and MCM3 in endometrial carcinogenesis. The authors investigated the immunohistochemical expression of MCM2 and MCM3, Ki-67, estrogen receptor, and progesterone receptor in 23 normal endometria, 9 endometrial hyperplasias, and 60 endometrial carcinomas. In the normal endometrial glands, the expression of MCM2 and MCM3 was significantly higher in the proliferative phase than in the secretory phase and was strongly correlated with Ki-67 expression. Similar correlation between the expression of MCMs and Ki-67 was also found in endometrial hyperplasia. In endometrial carcinomas, however, the expression of MCM2 and MCM3 was significantly lower than that in the normal proliferative endometrium. There was only a weak correlation between MCM2 and Ki-67, and no significant correlation between MCM3 and Ki-67 expression. These findings suggest that the expression of MCM2 and MCM3 directly reflects cell proliferation in normal and hyperplastic endometria. In endometrial carcinomas, however, there is a discrepancy between the expression of MCMs and cell proliferation, suggesting that the replication-licensing system may be aberrant in endometrial carcinomas.

To examine the sex steroid-dependent growth mechanisms of the human endometrium, the expression of steroid receptor coactivators [steroid receptor coactivator-1 (SRC-1) and p300/CREB-binding protein (p300/CBP)] and corepressors (nuclear receptor corepressor and silencing mediator for retinoid and thyroid hormone receptors) was examined by immunohistochemistry, using 50 samples of normal endometria, and was compared with that of estrogen receptors (ER), progesterone receptors (PR), and proliferation marker Ki-67. In addition, actual binding of the coactivators to ER or PR was analyzed by immunoprecipitation. The expression of SRC-1 was diffusely observed in glandular and stromal cells in the proliferative phase and drastically decreased in the secretory phase. Such change in the expression pattern of SRC-1 resembled that of ER, PR, and Ki-67. On the other hand, p300/CBP expression was relatively constant throughout the menstrual cycle, with slight predominance in the proliferative phase. The expression of corepressors nuclear receptor corepressor and silencing mediator for retinoid and thyroid hormone receptors was focal in the endometrium. Immunoprecipitation, using tissue samples of both proliferative and secretory phases, revealed the complex formation between the coactivators and receptors. Binding of SRC-1 to ER was observed in the proliferative (but not in the secretory) endometrium. In contrast, binding p300/CBP to ER was noted in the endometria of both phases. Complex formation between p300/CBP and PR was noted in the secretory endometrium, whereas that between SRC-1 and PR was not apparent. Accordingly, we showed the expression pattern of steroid receptor coactivators and corepressors in the normal endometrium. Cyclic change in the expression of SRC-1 during the menstrual cycle might be important in the estrogen-action for the glandular and stromal cells.

Brain metastases from endometrial carcinomas are rare and the treatment is usually difficult. Here, we report a patient with stage IV endometrial carcinoma whose brain metastases showed complete remission after stereotactic radiosurgery using a gamma-knife. A 48-year-old woman underwent removal of a single brain metastatic lesion, and one month later underwent hysterectomy for endometrioid-type G3, endometrial adenocarcinoma. After hysterectomy, a cranial magnetic resonance imaging (MRI) demonstrated multiple brain metastases and the patient received two courses of stereotactic radiosurgery and six courses of chemotherapy. Complete response of the brain lesions was obtained, and she is well without recurrence 38 months after the second stereotactic radiosurgery.

STUDY QUESTION How are protamine deficiencies associated with sperm head morphology in subfertile men? SUMMARY ANSWER The prevalence of morphological variations and large nuclear vacuoles was slightly higher in protamine-deficient spermatozoa than in non-deficient spermatozoa. WHAT IS KNOWN ALREADY A protamine deficiency was previously reported to be associated with an abnormal sperm morphology; however, how they are related to each other remains unclear. This is further confounded by a number of protamine-deficient spermatozoa having a normal head morphology. STUDY DESIGN, SIZE, DURATION This is a cross-sectional study, including 36 men diagnosed with male factor infertility or participating in an assisted reproduction program. To assess sperm head morphology, this study analyzed 2400 spermatozoa with a protamine deficiency and 2400 spermatozoa with a normal protamine status. An additional 21 men were analyzed to examine DNA fragmentation and its relationship with protamine deficiencies and sperm head morphologies. PARTICIPANTS/MATERIALS, SETTING, METHODS The morphology of the sperm head was evaluated based on its shape, size and nuclear vacuoles at a magnification of >6000×. Using elliptic Fourier analysis, the shape was summarized into four numeric variables. The protamine status was evaluated with chromomycin A3 (CMA3). Sperm head size, vacuoles and shape were compared between protamine-deficient and non-deficient spermatozoa. DNA fragmentation was evaluated with the terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assay. The percentages of protamine-deficient spermatozoa and DNA fragmentation were compared between spermatozoa with morphologically normal heads and those with abnormal heads. MAIN RESULTS AND THE ROLE OF CHANCE Variations in head size (P < 0.0001) and shape (P < 0.0001) were significantly higher, with narrower (P < 0.001), more fan-shaped (P < 0.01) and more square-shaped forms (P < 0.001) in protamine-deficient spermatozoa than in non-deficient spermatozoa; however, the distribution of morphological variations markedly overlapped. Protamine deficiencies were more frequently observed in spermatozoa with large nuclear vacuoles than in those without them (32.0 ± 3.1 versus 39.4 ± 2.9%, P < 0.001). The percentage of protamine-deficient spermatozoa was significantly lower in spermatozoa with a normal head morphology than in those with an abnormal head morphology (25.4 ± 2.6 versus 38.0 ± 2.5%, P < 0.001). The percentage of DNA fragmentation was significantly higher in protamine-deficient spermatozoa than in non-deficient spermatozoa (11.3 ± 2.1 versus 1.6 ± 0.6%, P < 0.001), and was lower in spermatozoa with a normal head morphology than in those with an abnormal head morphology (2.6 ± 0.7 versus 6.4 ± 0.2%, P < 0.001). LIMITATIONS, REASONS FOR CAUTION We were unable to discriminate the kind of protamines or quantify the extent of the protamine deficiency in spermatozoa using the CMA3 staining method. WIDER IMPLICATIONS OF THE FINDINGS This study provided a novel insight into how abnormal protamination affects sperm head morphology as well as the relationship between sperm head morphology and its own molecular integrity. Our results will contribute to a deeper understanding of the benefits and limitations of the morphological selection of spermatozoa for ICSI. STUDY FUNDING/COMPETING INTERESTS This study was supported by a JSPS Grant-in-Aid for the Encouragement of Scientists (25931009, 26931010). All authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER N/A.

Uterine leiomyosarcoma (Ut-LMS) more frequently develops in the muscle tissue layer of the uterine body than in the uterine cervix. The development of gynecological tumors is often correlated with the secretion of female hormones; however, the development of Ut-LMS has not been correlated with hormonal conditions, and risk factors remain unknown. A diagnostic biomarker that can distinguish malignant Ut-LMS from benign tumor leiomyoma (LMA) has not yet been established. Therefore, an analysis of the risk factors associated with human Ut-LMS is needed in order to establish a treatment method. Proteasome LMP2/β1i-deficient mice spontaneously develop Ut-LMS, with a disease prevalence of ~40% by 14 months of age. We found that the expression of LMP2/β1i was absent in human Ut-LMS, but present in human LMA. Therefore, the defective expression of LMP2/β1i may be one of the risk factors for Ut-LMS. LMP2/β1i is may be a potential diagnostic biomarker for human Ut-LMS, and may be a targeted molecule for a new therapeutic approach.

Progestins are known to suppress the growth of normal human endometrial glands and endometrial carcinomas possessing PRs. To elucidate the molecular mechanisms of progestin-induced growth inhibition, the expression and functional involvement of p27Kip1 (p27), a cyclin-dependent-kinase inhibitor, was investigated using cultured normal endometrial glandular cells and endometrial carcinoma cell lines (Ishikawa; PR-positive, KLE; PR-negative). Growth of the normal endometrial glandular cells and Ishikawa cells was suppressed by treatment with progesterone and medroxyprogesterone acetate, respectively, in association with an increase in p27 protein expression. Immunoprecipitation revealed that progestins accelerated the complex formation of p27 and cdk2 in both types of cells. However, treatment with progestins did not show any marked alterations in the mRNA expression of p27 in either normal glandular cells or Ishikawa cells. On the other hand, p27 protein degradation experiments indicated that treatment with progesterone and medroxyprogesterone acetate prolonged the degradation time of the normal endometrial glandular cells and Ishikawa cells, respectively. Forced expression of the p27 protein using a p27 expression plasmid reduced the growth activity of normal endometrial glandular cells. These findings suggest that p27 is functionally involved in progestin-induced growth suppression of normal and malignant endometrial epithelial cells and that up-regulation of the p27 protein by progestins possibly occurs via posttranslational mechanisms.

Cyclins are essential proteins in cell cycle control, and their deranged expression has been reported to be associated with malignant transformation. Involvement of cyclins in the development of endometrioid carcinomas of the endometrium was studied immunohistochemically using antibodies against both cyclin A and tumor suppressor gene product p53, and their expression was compared with that of Ki-67 antigen. Sixty-two cases of endometrial endometrioid carcinoma and 20 cases of normal endometrium (10 proliferative and 10 secretory phase) were examined. Of the 62 endometrioid carcinomas, atrophic endometrium and hyperplasia were found adjacent to the cancers in 30 and 19 cases respectively. Cyclin A was expressed in1% of the glandular cells of normal endometrium in the proliferative phase and in hyperplasia, but was negligible in normal secretory phase and atrophic endometrium. p53 was almost always negative in normal endometrium and hyperplasia. Of the 62 endometrioid carcinomas, 12 tumors (19.4%) overexpressed cyclin A and 21 tumors (33.8%) overexpressed p53 (positive cells1%). Cyclin A and p53 were more frequently expressed in poorly differentiated tumors than in well differentiated tumors (cyclin A, p = 0.002; p53, p = 0.016). In addition, cyclin A-positive cells were topographically related to those cells positive for p53 as well as Ki-67. In conclusion, the abnormal expression of cyclin A and p53 is associated with high-grade endometrial endometrioid carcinomas.

BACKGROUND Mucinous tumors of the ovary have been thought to originate in two ways: by mullerian-type metaplasia of surface epithelium, and as monodermal teratomas. To gain a better understanding of their pathogenesis, the authors analyzed these tumors for their expression of estrogen receptors (ER) and progesterone receptors (PR) as markers of mullerian-type differentiation and for their content of gastric-type mucin as a marker of gastric differentiation. METHODS The histochemical expression of ER, PR, and gastric-type mucin was studied in 10 specimens of the cervix with normal endocervical glands (as a representative of mullerian-derived mucin-containing cells), 3 ovary specimens with surface epithelial inclusion cysts that contained endocervical-like mucin-containing cells (representing mullerian-type metaplasia), and 47 mucinous tumors of the ovary (29 benign, 8 with low malignant potential, and 10 malignant). RESULTS Normal endocervical glands expressed ER and PR and rarely expressed gastric-type mucin. Ovarian inclusion cysts showed strong expression of ER and PR in the cuboidal cells and drastically reduced expression in the endocervical-like mucin-containing cells. The cuboidal cells were negative for gastric-type mucin, but the endocervical-like mucin-containing cells expressed gastric-type mucin. Endocervical-like mucinous cells in benign and borderline mucinous tumors showed expression of PR and/or gastric-type mucin in all cases. CONCLUSIONS The staining results for the inclusion cysts support the thesis that the endocervical-like mucinous cells encountered in the ones that express ER and PR weakly or not at all and have histochemical properties of normal gastric epithelium have their origin in metaplasia of mullerian-type epithelium. Application of the same staining methods to benign ovarian tumors and those with low malignant potential suggests strongly that similar mullerian-type metaplasia is a major pathway in their pathogenesis. Cancer 1997; 80:908-16. © 1997 American Cancer Society.

The invasion of extravillous trophoblasts (EVTs) to the decidua and spiral arteries in early pregnancy is a crucial step for a successful pregnancy; however, its mechanisms are not fully understood. Lipocalin2 (LCN2), a multifunctional secretory protein known as neutrophil gelatinase-associated lipocalin (NGAL), reportedly enhanced invasiveness via the activation of matrix metalloproteinase-9 (MMP-9) in several cancer cells. In this study, the expression and function of LCN2 in early placenta were analyzed.Early placental tissues between 7 and 10 weeks of gestation were obtained from normal pregnant women who underwent elective termination. The expression of LCN2 was examined using immunostaining and RT-PCR. EVTs isolated from these placental tissues and a choriocarcinoma cell line (JAR) were used to investigate the effects of LCN2 on proliferation, invasion potential, and MMP-9 activity under hypoxia using a WST-1 assay, Matrigel invasion assay, and gelatin gel zymography, respectively.The immunohistochemical expression of LCN2 was observed in the cytoplasm of EVTs, cytotrophoblasts and the decidua, but not in syncytiotrophoblasts. The addition of recombinant LCN2 did not affect proliferation, but enhanced the invasiveness (500 ng/mL, p0.01) and MMP-9 activity of primary cultured EVTs and JAR in a dose-dependent manner. Silencing LCN2 using shRNA reduced the invasiveness (p0.01) and MMP-9 activity of JAR. In addition, the hypoxic condition (2% O₂) increased LCN2 expression (p0.01), MMP-9 activity, and invasive ability (p0.01).LCN2 was involved in the invasiveness of EVTs, especially under hypoxia, via increased MMP-9 activity.

Polypoid endometriosis is a rare type of endometriosis. We report a case of polypoid endometriosis of the ovary mimicking ovarian carcinoma with peritoneal dissemination. Computed tomography and magnetic resonance imaging showed a left ovarian endometriotic cyst containing several nodules in the cystic wall that displayed enhancement, and pelvic nodules on the right ovary. A preoperative or intraoperative diagnosis to avoid the unnecessary extended operation is important for such disease. Retrospective magnetic resonance imaging analysis identified a peculiar finding for polypoid endometriosis: all solid nodules had a round and smooth shape and displayed a low-signal-intense marginal edge on T2-weighted images, suggesting that this is an important finding for differentiating polypoid endometriosis from ovarian carcinoma arising from endometriosis.

BACKGROUND In the normal cell cycle, tumor suppressor gene products (p53) and cyclin (cyclin D1) cooperate. Abnormalities in the cooperation of these factors may result in malignant transformation of the cell. Mutant p53 protein overexpression is defined in many human cancers, including endometrial carcinoma. This study investigated the role of cyclin D1 in the development of human uterine endometrial carcinoma. METHODS Seventy-four patients whose pathology slides contained either normal or hyperplastic endometrium adjacent to endometrial carcinoma were studied. Immunohistochemical staining of the serial paraffin sections was performed using antibodies to p53 and cyclin D1. RESULTS The expression of cyclin D1 was restricted to only a few cells of normal and hyperplastic endometrium, whereas it was preferentially expressed in 40% (30/74) of endometrial carcinomas. The cells that overexpressed cyclin D1 also overexpressed p53. Moreover, all 30 cases with varied distributions of cyclin D1-positive cells corresponded identically with the distribution of p53-positive cells. Diffuse positivity for cyclin D1 was specifically observed in clinically advanced stages of pathologic G2 and G3 tumors. CONCLUSIONS The data suggest that coabnormal expression of cyclin D1 and p53 protein may contribute to the development of endometrial carcinoma and may also be involved in the progression to malignancy. Cancer 1996;78:1248-53.

BACKGROUND In the normal cell cycle, the appropriate interaction of factors such as tumor suppressor gene products (retinoblastoma susceptibility [Rb], p53) and cyclins is essential. Abnormalities in the interaction of these factors may result in malignant transformation of the cell. Malignant transformation of the endometrium, which is believed to be a sex steroid-dependent tumor, probably involves a process of uncoupling of these factors and sex steroid hormone receptors. This study is designed to test this hypothesis. METHODS Fifty-six patients whose pathology slides contained either normal or hyperplastic endometrium adjacent to endometrial carcinoma were selected. Immunohistochemical staining of serial paraffin sections was performed using antibodies to estrogen receptors (ER) and progesterone receptors (PR), p53, and Rb, as well as cyclin E. RESULTS The normal and hyperplastic endometria adjacent to carcinoma showed positive staining for ER and PR and negative staining for p53. Of 56 carcinomas, 39 (69.6%) showed homogeneous positive staining for ER and PR and negative staining for p53, whereas the remaining 17 carcinomas (30.4%) contained varied distributions of ER- and PR-negative cells, and p53-positive cells (6 were negative or focally positive for ER/PR and diffusely-positive for p53, 4 were regionally-positive for ER/PR and regionally-positive for p53, and 7 were diffusely positive for ER/PR and focally-positive for p53). The p53-positive cells corresponded to those that stained negatively for ER/PR. This topographic inverse relationship between ER/PR expression and p53 expression also correlated with the staining intensities. Furthermore, the cells with weak or negative staining for p53 had a tendency to stain positively for Rb and weakly positive for cyclin E, whereas the cancer cells with definite positivity for p53 tended to stain either weakly or negatively for Rb and definitely positive for cyclin E. The cells showing diffusely positive for p53 were present significantly in clinical Stage III and pathologic Grade G3. CONCLUSIONS In the development of endometrial carcinoma, stepwise abnormalities of sex steroid receptors, tumor suppressor gene products, and cyclins apparently exist, and may correlate with the progression of the malignant process. Cancer 1996;77:321-9.

Cell cycle regulatory molecules were analysed in normal human endometrial tissue using antibodies against cyclins D1, E, A, and B1 and cyclin-dependent kinases (CDKs) cdk4, cdk2, and cdc2. The expression of these regulatory molecules in gland cells and stromal cells was compared with the expression of oestrogen receptors (ER), progesterone receptors (PR), and Ki67 (a growth-related molecule). In general, a substantially higher percentage of the gland cells stained positive for cyclins and CDKs during the proliferative phase of the menstrual cycle. Cyclin E, cdk2 and/or cdk4 were especially apparent in the cytoplasm of most of the gland cells as well as in the stromal cells. In contrast, most of the regulatory molecules were undetectable in the gland cells by the end of the secretory phase of the cycle, but they did not decline in the stromal cells. The data also revealed that ER, PR, and Ki67 in both gland cells and stromal cells follow the same basic pattern of expression as the cyclins and CDKs. These results suggest that cyclins and CDKs are functionally involved in the rhythmic proliferation of normal human endometrial tissue, and the action of these agents may be related to the endometrial levels of sex steroids and Ki67.