||Yagi H¹, Onoyama I¹, Asanoma K¹, Hori E¹, Yasunaga M¹, Kodama K¹, Kijima M¹, Ohgami T¹, Kaneki E¹, Okugawa K¹, Yahata H¹, Kato K¹, Gα13-mediated LATS1 down-regulation contributes to epithelial-mesenchymal transition in ovarian cancer., FASEB J, doi: 10.1096/fj.201901278R, Epub ahead of print, 2019.09, Gα13, a heterotrimeric G-protein of the Gα12/13 subfamily, is associated with aggressive phenotypes in various human cancers. However, the mechanisms by which Gα13 promotes cancer progression have not been fully elucidated. Here, we demonstrate that the activation of Gα13 induces epithelial-mesenchymal transition in ovarian cancer (OvCa) cells through down-regulation of large tumor suppressor kinase (LATS) 1, a critical component of the Hippo signaling pathway. A synthetic biology approach using a mutant GPCR and chimeric G-protein revealed that Gα13-regulated phosphorylation of LATS1 at serine 909 within its activation loop induced recruitment of the itchy E3 ubiquitin protein ligase to trigger LATS1 degradation. Our findings uncover novel mechanisms through which Gα13 activation induces dysregulation of the Hippo signaling pathway, which leads to aggressive cancer phenotypes, and thereby identify a potential target for preventing the metastatic spread of OvCa.-Yagi, H., Onoyama, I., Asanoma, K., Hori, E., Yasunaga, M., Kodama, K., Kijima, M., Ohgami, T., Kaneki, E., Okugawa, K., Yahata, H., Kato, K. Gα13-mediated LATS1 down-regulation contributes to epithelial-mesenchymal transition in ovarian cancer..
||Yahata H¹, Sonoda K¹, Okugawa K¹, Yagi H¹, Ohgami T¹, Yasunaga M¹, Onoyama I¹, Kaneki E¹, Asanoma K¹, Kato K¹, Survey of the desire to have children and engage in sexual activity after trachelectomy among young Japanese women with early-stage cervical cancer, J Obstet Gynaecol Res, 10.1111/jog.14099, 2019.08, AIM: To evaluate how the desire to have children and engage in sexual activity change after trachelectomy in Japanese women with early-stage cervical cancer who strongly desired to have children before surgery.
METHODS: Desire to have children, coital pain, fear of sexual intercourse, sexual activity frequency and libido were assessed in cervical cancer patients who received follow-up after trachelectomy. An anonymous questionnaire survey was conducted via informed consent.
RESULTS: Of the 151 patients who underwent trachelectomy at Kyushu University Hospital between 2005 and 2015, 46 patients were evaluated; the response rate was 30%. The desire to have children disappeared in 13 of 46 (28%) patients, and 14 (30%) patients experienced increased coital pain. Moreover, 19 (41%) patients experienced fear of sexual intercourse, and sexual frequency decreased in 24 (52%) patients.
CONCLUSION: Trachelectomy is an important fertility-sparing surgical method; however, this study revealed loss of the desire to have children and/or to engage in sexual activity in some patients after surgery. Counseling about these issues is important and should be addressed..
||Yahata H, Kobayashi H, Sonoda K, Kodama K, Yagi H, Yasunaga M, Ohgami T, Onoyama I, Kaneki E, Okugawa K, Baba S, Isoda T, Ohishi Y, Oda Y, Kato K, Prognostic outcome and complications of sentinel lymph node navigation surgery for early-stage cervical cancer., Int J Clin Oncol, 10.1007/s10147-018-1327-y, 2018.08, BACKGROUND: To evaluate the prognostic outcome and surgical complications in patients with early-stage cervical cancer who underwent sentinel node navigation surgery (SNNS) for hysterectomy or trachelectomy.
METHODS: A total of 139 patients who underwent SNNS using 99mTc phytate between 2009 and 2015 were evaluated. No further lymph node dissection was performed when intraoperative analysis of the sentinel lymph nodes (SLNs) was negative for metastasis. We compared the surgical complications between the SNNS group and 67 matched patients who underwent pelvic lymph node dissection (PLND) after SLN mapping between 2003 and 2008. We also examined the clinical outcomes in the SNNS group.
RESULTS: The mean number of detected SLNs was 2.5 per patient. Fourteen of the 139 patients in the SNNS group underwent PLND based on the intraoperative SLN results. The amount of blood loss, the operative time, and the number of perioperative complications were significantly less in the SNNS group than in the matched PLND group. There was no recurrence during a follow-up period ranging from 2 to 88 months (median 40 months) in the SNNS group.
CONCLUSIONS: Using SNNS for early-stage cervical cancer is safe and effective and does not increase the recurrence rate. A future multicenter trial is warranted..
||Yasutake N¹, Ohishi Y¹, Taguchi K², Hiraki Y³, Oya M³, Oshiro Y⁴, Mine M⁵, Iwasaki T¹, Yamamoto H¹, Kohashi K¹, Sonoda K⁶, Kato K⁶, Oda Y¹, Insulin-like growth factor II messenger RNA-binding protein-3 is an independ-ent prognostic factor in uterine leiomyosarcoma, Histopathology, 10.1111/his.13422, 72, 5, 739-748, 2018.04, AIMS: The aim of this study was to identify the prognostic factors of uterine leiomyosarcoma (ULMS).
METHODS AND RESULTS: We reviewed 60 cases of surgically resected ULMSs and investigated conventional clinicopathological factors, together with the expression of insulin-like growth factor II messenger RNA-binding protein-3 (IMP3), hormone receptors and cell cycle regulatory markers by immunohistochemistry. Mediator complex subunit 12 (MED12) mutation analysis was also performed. Univariate analyses revealed that advanced stage (P < 0.0001), older age (P = 0.0244) and IMP3 expression (P = 0.0011) were significant predictors of a poor outcome. Multivariate analysis revealed advanced stage (P < 0.0001) and IMP3 (P = 0.0373) as independent predictors of a poor prognosis. Expressions of cell cycle markers and hormone receptors, and MED12 mutations (12% in ULMSs) were not identified as prognostic markers in this study.
CONCLUSIONS: IMP3 expression in ULMS could be a marker of a poor prognosis..
||Yahata H, Sonoda K, Yasunaga M, Ogami T, Kawano Y, Kaneki E, Okugawa K, Tsunehisa Kaku, Kato K, Surgical treatment and outcome of early invasive adenocarcinoma of the uterine cervix (FIGO stage IA1)., Asia Pac J Clin Oncol 2017, 10.1111/ajco.12691, 14, 2, e50-e53, 2018.04, AIM: To investigate the surgical outcome of FIGO stage IA1 cervical adenocarcinoma.
METHODS: Between 2005 and 2011, 12 patients from Kyushu University Hospital had cervical adenocarcinoma, with a tumor depth of less than 3 mm and a horizontal width of less than 7 mm (FIGO stage IA1), diagnosed by cervical conization. All patients underwent simple hysterectomy or simple trachelectomy with pelvic lymphadenectomy.
RESULTS: The mean patient age was 34 years (range, 26-70 years). The median follow-up period was 70.5 months (range, 26-99 months). No pelvic lymph-node metastasis was seen, and no patient experienced disease recurrence.
CONCLUSION:Early invasive cervical adenocarcinoma with a depth of invasion of 3 mm or less and a horizontal spread of 7 mm or less has little potential for nodal metastasis or recurrence. Therefore, simple hysterectomy or trachelectomy, without lymphadenectomy, might be an alternative treatment option for stage IA1 cervical adenocarcinoma..
||Ohmaru-Nakanishi T¹, Asanoma K², Fujikawa M¹, Fujita Y¹, Yagi H¹, Onoyama I¹, Hidaka N¹, Sonoda K¹, Kato K¹, Fibrosis in Preeclamptic Placentas Is Associated with Stromal Fibroblasts Acti-vated by the Transforming Growth Factor Beta 1 (TGFB1) Signaling Pathway., Am J Pathol, 10.1016/j.ajpath.2017.11.008, 188, 3, 683-695, 2018.03, Although fibrosis is one of the most prominent pathologic features of preeclamptic (PE) placentas, its mechanism remains largely unknown. Consistent with previous reports, we observed overexpression of collagen; actin, α2, smooth muscle, aorta; connective tissue growth factor; and fibronectin in PE placentas compared with control ones. To investigate the mechanism of fibrosis in PE placentas, placental fibroblasts were isolated from PE placentas or normal pregnancies at delivery. The expression of fibrosis-related factors in fibroblasts was evaluated by real-time RT-PCR, Western blotting, enzyme-linked immunosorbent assay, and gene microarrays. An in vitro collagen gel contraction assay was also performed. Fibroblasts isolated from PE placentas showed higher expression levels of fibrosis-related factors compared with those from control ones. Global gene expression profiling of PE fibroblasts was contrasted with that of control ones and indicated an intimate association with transforming growth factor-β1 (TGF-β1) signaling. Furthermore, the PE fibroblasts expressed abundant phosphorylated SMAD family member 2 and showed higher expression levels of target genes of TGF-β1 signaling compared with the control ones. The PE fibroblasts also had a greater ability to contract compared with the control ones. Contractility also depended on TGF-β1 signaling. Our results suggest that TGF-β1 signaling is activated in the fibroblasts in PE placentas and that these active fibroblasts contribute to fibrosis..
||Hidaka N , Kido S, Sato Y, Murata M, Fujita Y, Kato K, Thoracoamniotic shunting for fetal pleural effusion with hydropic change using a double-basket catheter: An insight into the preoperative determinants of shunt-ing efficacy., Eur J Obstet Gynecol Reprod Biol, 10.1016/j.ejogrb.2017.12.008, 221, 34-39, 2018.02, OBJECTIVES: Although the efficacy of thoracoamniotic shunting (TAS) for fetal hydrothorax is well-recognized, the coexistence of hydrops fetalis is still a clinical challenge. The preoperative determinants of shunting efficacy are not fully understood. In this study, we aimed to investigate the perinatal and postnatal outcomes of hydrops fetalis with pleural effusion treated by TAS using a double-basket catheter, and to discuss the preoperative factors predictive of patients who will benefit from TAS.
STUDY DESIGN: We conducted a retrospective study in hydropic fetuses with pleural effusion treated by TAS between 2007 and 2015. We extracted information regarding postnatal survival and pretherapeutic sonographic findings, including skin-edema thickness, pleural-effusion pocket size, and Doppler readings.
RESULTS: Twelve subjects underwent TAS at a median gestational age of 29+5 weeks (range, 25+5-33+2 weeks). Skin edema disappeared or regressed in 7. Three experienced early neonatal death and the other 9 ultimately survived after a live birth at a median gestational age of 33+4 weeks (range, 29+1-38+2 weeks). All surviving children, except for 1, had a pretherapeutic pleural-effusion pocket greater than the precordial-edema thickness. All 3 children that died had precordial-edema thickness equal to or greater than the size of the pleural-effusion pocket.
CONCLUSIONS: We achieved a high survival rate (75%) using the double-basket technique. A greater pretherapeutic width of skin edema compared with the pleural-effusion pocket is possibly suggestive of a treatment-resistant condition and subsequent poor postnatal outcome..
||Sonoda K¹, Yahata H, Okugawa K, Kaneki E, Ohgami T, Yasunaga M, Baba S, Oda Y, Honda H, Kato K, Value of Intraoperative Cytological and Pathological Sentinel Lymph Node Di-agnosis in Fertility-Sparing Trachelectomy for Early-Stage Cervical Cancer, Oncology, 10.1159/000484049, 94, 2, 92-98, 2018.02, BACKGROUND AND OBJECTIVES: Trachelectomy, a fertility-sparing surgery for early-stage cervical cancer, can be performed only when there is no extrauterine extension present. Therefore, identifying the sentinel lymph nodes (SLNs) and using them to obtain an intraoperative pathologic diagnosis can provide information on the feasibility and safety of trachelectomy. Our aim was to assess the value of an intraoperative SLN diagnosis.
METHODS: We retrospectively analyzed the accuracy of intraoperative imprint cytology and frozen-section examination in 201 patients at our institution in whom trachelectomy was planned.
RESULTS: All patients could be evaluated for SLNs; a total of 610 SLNs were analyzed. Although the specificity of both imprint cytology and frozen-section examination was 100.0%, the sensitivity was only 58.6 and 65.5%, respectively. The diagnostic sensitivity was higher in 2-mm slices along the short axis than on bisection along the longitudinal axis. Imprint cytology correctly diagnosed 2 patients who had false-negative results on frozen section. The nature of the metastatic foci that caused an intraoperative false-negative diagnosis was either micrometastasis or isolated tumor cells.
CONCLUSIONS: The accuracy of intraoperative SLN diagnosis requires improvement, especially when small metastatic foci are present..
||Morokuma S, Michikawa T, Yamazaki S, Nitta H, Kato K, Association between exposure to air pollution during pregnancy and false posi-tives in fetal heart rate monitoring., Scientific reports, 10.1038/s41598-017-12663-2, 7, 1, 1-8, 2017.09, Fetal heart rate (FHR) monitoring is essential for fetal management during pregnancy and delivery but results in many false-positive diagnoses. Air pollution affects the uterine environment; thus, air pollution may change FHR reactivity. This study assessed the association between exposure to air pollution during pregnancy and FHR monitoring abnormalities using 2005-2010 data from the Japan Perinatal Registry Network database. Participants were 23,782 singleton pregnant women with FHR monitoring, without acidemia or fetal asphyxia. We assessed exposure to air pollutants, including particulate matter (PM), ozone, nitrogen dioxide (NO2), and sulfur dioxide (SO2). In a multi-trimester model, first-trimester PM exposure was associated with false positives in FHR monitoring (odds ratio [OR] per interquartile range (10.7 μg/m3) increase = 1.20; 95% CI: 1.05-1.37), but not second-trimester exposure (OR = 1.05; 95% CI: 0.91-1.21) and third-trimester exposure (OR = 1.06; 95% CI: 0.96-1.17). The association with first-trimester PM exposure persisted after adjustment for exposure to ozone, NO2, and SO2; however, ozone, NO2, and SO2 exposure was not associated with false positives in FHR monitoring. First-trimester PM exposure may alter fetal cardiac response and lead to false positives in FHR monitoring..
||Kido S, Hidaka N, Sato Y, Fujita Y, Miyoshi K, Nagata K, Taguchi T, Kato K, Re-evaluation of lung to thorax transverse area ratio immediately before birth in predicting postnatal short-term outcomes of fetuses with isolated left-sided congenital diaphragmatic hernia: a single center analysis., Congenit Anom (Kyoto), 10.1111/cga.12243, 1-6, 2017.08, We aimed to investigate whether the lung-to-thorax transverse area ratio (LTR) immediately before birth is of diagnostic value for the prediction of postnatal short-term outcomes in cases of isolated left-sided congenital diaphragmatic hernia (CDH). We retrospectively reviewed the cases of fetal isolated left-sided CDH managed at our institution between April 2008 and July 2016. We divided the patients into two groups based on LTR immediately before birth, using a cut-off value of 0.08. We compared the proportions of subjects within the two groups who survived until discharge using Fisher's exact test. Further, using Spearman's rank correlation, we assessed whether LTR was correlated with length of stay, duration of mechanical ventilation, and supplemental oxygen. Twenty-nine subjects were included (five with LTR < 0.08, and 24 with LTR ≥ 0.08). The proportion of subjects surviving until discharge was 40% (2/5) for patients with LTR < 0.08, as compared with 96% (23/24) for those with LTR ≥ 0.08. LTR measured immediately before birth was negatively correlated with the postnatal length of stay (Spearman's rank correlation coefficient, rs = -0.486), and the duration of supplemental oxygen (rs = -0.537). Further, the duration of mechanical ventilation was longer in patients with a lower LTR value. LTR immediately before birth is useful for the prediction of postnatal short-term outcomes in fetuses with isolated left-sided CDH. In particular, patients with prenatal LTR value less than 0.08 are at increased risk of postnatal death..
||Okawa H, Morokuma S, Maehara K, Arata A, Ohmura Y, Horinouchi T, Konishi Y, Kato K, Eye movement activity in normal human fetuses between 24 and 39 weeks of gestation., PLos One, 10.1371/journal.pone.0178722, 12, 7, 1-12, 2017.07, Rapid eye movement (REM) sleep occurs throughout a relatively large proportion of early development, and normal REM activity appears to be required for healthy brain development. The eye movements (EMs) observed during REM sleep are the most distinctive characteristics of this state. EMs are used as an index of neurological function postnatally, but no specific indices of EM activity exist for fetuses. We aimed to identify and characterize EM activity, particularly EM bursts suggestive of REM periods, in fetuses with a gestational age between 24 and 39 weeks. This cross-sectional study included 84 normal singleton pregnancies. Fetal EMs were monitored using real-time ultrasonography for 60 min and recorded as videos. The videos were manually converted into a time series of EM events, which were then analyzed by piecewise linear regression for various EM characteristics, including EM density, EM burst density, density of EMs in EM bursts, and continuous EM burst time. Two critical points for EM density, EM burst density, and density of EMs in EM bursts were evident at gestation weeks 28-29 and 36-37. Overall EM activity in human fetuses increased until 28-29 weeks of gestation, then again from 36-37 to 38-39 weeks of gestation. These findings may be useful for creating indices of fetal neurological function for prognostic purposes..
||Okugawa K, Kobayashi H, Sonoda K, Kaneki E, Kawano Y, Hidaka N, Egashira K, Fujita Y, Yahata H, Kato K, Oncologic and obstetric outcomes and complications during pregnancy after fertility-sparing abdominal trachelectomy for cervical cancer:a retrospective review.
, Int J Oncol, 10.1007/s10147-016-1059-9, 22, 2, 340-346, 2017.04, BACKGROUND: Trachelectomy was developed as a fertility-sparing surgery for early-stage cervical cancer in patients of childbearing age. The purpose of this study is to evaluate oncologic and obstetric outcomes and complications after abdominal trachelectomy.
METHODS: We began to perform abdominal trachelectomy in 2005. Our institutional review board approved this clinical study, and fully informed consent was obtained from each patient. The medical records of patients who underwent trachelectomy were retrospectively reviewed.
RESULTS: We performed 151 abdominal trachelectomies (89 radical trachelectomies, 48 modified radical trachelectomies, and 14 simple trachelectomies). The median age of the patients was 33 years, and the median postoperative follow-up period was 61 months. Although one patient experienced recurrence at the preserved cervix, none died after treatment. A total of 61 patients attempted to conceive after trachelectomy, and 21 pregnancies were achieved in 15 women. Hence, the pregnancy rate among patients who attempted to conceive was 25%. Fifteen babies were delivered by cesarean section between gestational weeks 23 and 37. Six babies were delivered at term. Six cases of preterm premature rupture of the membranes occurred. Varices appeared around the uterovaginal anastomotic site in five patients.
CONCLUSIONS: Our data indicate that the oncologic outcome was excellent but infertility treatment was necessary to achieve the majority of conceptions. Additionally, preterm premature rupture of the membranes and premature delivery were frequently observed. An improved pregnancy rate and prevention of complications during pregnancy are issues that should be addressed in future studies.
||Morokuma S, Tsukimori K, Hori T, Kato K, Furue M, The Vernix Caseosa is the Main Site of Dioxin Excretion in the Human Foetus., Sci Rep, 10.1038/s41598-017-00863-9, 7, 1, 739-739, 2017.04, Dioxins are highly toxic to foetuses and prenatal exposure leads to adverse health effects; however, the metabolic pathways involved in dioxin excretion are poorly understood. We determined the dynamics of maternal-to-foetal dioxin transfer during normal pregnancy and how foetuses eliminate polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans, and non-ortho polychlorinated biphenyls. Dioxin levels in maternal blood, cord blood, placenta, vernix caseosa, meconium, and amniotic fluid were analysed by high-resolution gas chromatography/mass spectrometry. The average levels of total dioxins, expressed as picograms of toxic equivalency quantity per gram of lipid and in parentheses, dioxin fraction, with maternal blood levels arbitrarily set as 100%, were as follows: maternal blood, 15.8 (100%); placenta, 12.9 (81.5%); cord blood, 5.9 (37.2%); vernix caseosa, 8.4 (53.2%); meconium, 2.9 (18.2%); and amniotic fluid, 1.5 (9.2%). Similar proportions were observed for each dioxin congener. Thus, the highest content of foetal dioxins was observed in the vernix caseosa, indicating that this is the major site of dioxin excretion in human foetuses..
||Kitade S, Onoyama I, Kobayashi H, Yagi H, Kato M, Tsunematsu R, Asanoma K, Sonoda K, Wake N, Hata K, Nakayama K, Kato K, FBXW7 is involved in the acquisition of the malignant phenotype in epithelial ovarian Tumors., Cancer Sci, 10.1111/cas.13026., Epub ahead of print, 2016.08, FBXW7 is a ubiquitin ligase that mediates ubiquitylation of oncoproteins, such as c-Myc, cyclin E, Notch and
c-Jun. FBXW7 is a known tumor-suppressor gene, and mutations in FBXW7 have been reported in various
human malignancies. In this study, we examined the sequences of the FBXW7 and p53 genes in 57 ovarian
cancer clinical samples. Interestingly, we found no FBXW7 mutations associated with amino acid changes.
We also investigated FBXW7 expression levels in 126 epithelial ovarian tumors. FBXW7 expression was
negatively correlated with the malignant potential of ovarian tumors. That is to say, FBXW7 expression
levels in ovarian cancer samples were significantly lower than those in borderline and benign tumors
(P < 0.01). FBXW7 expression levels in serous carcinoma samples were the lowest among four major
histological subtypes. In addition, p53-mutated ovarian cancer samples showed significantly lower levels
of FBXW7 expression compared with p53 wild-type cancer samples (P < 0.001). DNA methylation arrays
and bisulfite PCR sequencing experiments revealed that 5'-upstream regions of FBXW7 gene in p53-mutated
samples were significantly higher methylated compared with those in p53 wild-type samples (P < 0.01).
This data indicates that p53 mutations might suppress FBXW7 expression through DNA hypermethylation
of FBXW7 5'-upstream regions. Thus, FBXW7 expression was downregulated in ovarian cancers, and was
associated with p53 mutations and the DNA methylation status of the 5'-upstream regions of FBXW7..
||Yahata H, Kobayashi H, Sonoda K, Shimokawa M, Ogami T, Saito T, Ogawa S, Sakai K, Ichinoe A, Ueoka Y, Hasuo Y, Nishida M, Masuda S, Kato K, Efficacy of aprepitant for the prevention of chemotherapy-induced nausea and vomiting with a moderately
emetogenic chemotherapy regimen: a multicenter, placebo-controlled, double-blind, randomized study in
patients with gynecologic cancer receiving paclitaxel and carboplatin., Int J Clin Oncol, 10.1007/s10147-015-0928-y., 21, 3, 491-497, 2016.06, BACKGROUND: Substance P contributes to the hypersensitivity reaction (HSR) to paclitaxel in a rat model.
Aprepitant acts as an inhibitor of the binding of substance P to the neurokinin-1 receptor and, consequently,
may reduce the frequency of paclitaxel-induced HSR. While aprepitant has a prophylactic effect against
vomiting caused by high-dose cisplatin, the benefits of aprepitant have not been clearly demonstrated in
patients receiving paclitaxel and carboplatin (TC) combination chemotherapy.
METHODS: We conducted a multicenter, placebo-controlled, double-blind, randomized study in Japanese
patients with gynecologic cancer who received TC combination chemotherapy. Patients received aprepitant
or placebo together with both a 5-HT3 receptor antagonist and dexamethasone prior to chemotherapy.
The primary endpoint was the proportion of patients with HSR, and the secondary endpoints were the
proportion of patients with "no vomiting", "no significant nausea", and complete response, respectively.
RESULTS: Of the 324 randomized patients, 297 (151 in the aprepitant group; 146 in the placebo group)
were evaluated. The percentage of patients with HSR (9.2 vs. 7.5 %, respectively; P = 0.339) was not
significantly different between the groups. The percentage of "no vomiting" patients (78.2 vs. 54.8 %;
P < 0.0001), "no significant nausea" patients (85.4 vs. 74.7 %; P = 0.014), and patients showing complete
response (61.6 vs. 47.3 %, P = 0.0073) was significantly higher in the aprepitant group than in the placebo
CONCLUSION: The administration of aprepitant did not have a prophylactic effect on the HSR but was
effective in reducing nausea and vomiting in gynecologic cancer patients receiving TC combination
||Masuda A, Katoh N, Nakabayashi K, Kato K, Sonoda K, Kitade M, Takeda S, Hata K, Tomikawa J, An improved method for isolation of epithelial and stromal cells from the human endometrium., The Journal of reproduction and development, 10.1262/jrd.2015-137, 62, 2, 213-218, 2016.04, We aimed to improve the efficiency of isolating endometrial epithelial and stromal cells (EMECs and EMSCs) from the human endometrium. We revealed by immunohistochemical staining that the large tissue fragments remaining after collagenase treatment, which are usually discarded after the first filtration in the conventional protocol, consisted of glandular epithelial and stromal cells. Therefore, we established protease treatment and cell suspension conditions to dissociate single cells from the tissue fragments and isolated epithelial (EPCAM-positive) and stromal (CD13-positive) cells by fluorescence-activated cell sorting. Four independent experiments showed that, on average, 1.2 × 10(6) of EMECs and 2.8 × 10(6) EMSCs were isolated from one hysterectomy specimen. We confirmed that the isolated cells presented transcriptomic features highly similar to those of epithelial and stromal cells obtained by the conventional method. Our improved protocol facilitates future studies to better understand the molecular mechanisms underlying the dynamic changes of the endometrium during the menstrual cycle..
||Yagi H, Asanoma K, Ohgami T, Ichinoe A, Sonoda K, Kato K, GEP oncogene promotes cell proliferation through YAP activation in ovarian cancer., Oncogene, 10.1038/onc.2015.505., 2016.01, G-protein-coupled receptors (GPCRs) and their ligands function in the progression of human malignancies. Gα12 and Gα13, encoded by GNA12 and GNA13, respectively, are referred to as the GEP oncogene and are implicated in tumor progression. However, the molecular mechanisms by which Gα12/13 activation promotes cancer progression are not fully elucidated. Here, we demonstrate elevated expression of Gα12/13 in human ovarian cancer tissues. Gα12/13 activation did not promote cellular migration in the ovarian cancer cell lines examined. Rather, Gα12/13 activation promoted cell growth. We used a synthetic biology approach using chimeric G proteins and GPCRs activated solely by artificial ligands to selectively trigger signaling pathways downstream of specific G proteins. We found that Gα12/13 promotes proliferation of ovarian cancer cells by activating the transcriptional coactivator YAP, a critical component of the Hippo signaling pathway. Furthermore, we reveal that inhibition of YAP by short hairpin RNA or a specific inhibitor prevented the growth of ovarian cancer cells. Therefore, YAP may be a suitable therapeutic target in ovarian cancer.Oncogene advance online publication, 25 January 2016; doi:10.1038/onc.2015.505..
||Inagaki T, Kusunoki S, Tabu K, Okabe H, Yamada I, Taga T, Matsumoto A, Makino S, Takeda S, Kato K, Up-regulation of lymphocyte antigen 6 complex expression in side-population cells de-rived from a human trophoblast cell line HTR-8/SVneo., Human Cell, 10.1007/s13577-015-0121-7, 29, 1, 10-21, 2016.01, The continual proliferation and differentiation of trophoblasts are critical for the maintenance of pregnancy. It is well known that the tissue stem cells are associated with the development of tissues and pathologies. It has been demonstrated that side-population (SP) cells identified by fluorescence-activated cell sorting (FACS) are enriched with stem cells. The SP cells in HTR-8/SVneo cells derived from human primary trophoblast cells were isolated by FACS. HTR-8/SVneo-SP cell cultures generated both SP and non-SP (NSP) subpopulations. In contrast, NSP cell cultures produced NSP cells and failed to produce SP cells. These SP cells showed self-renewal capability by serial colony-forming assay. Microarray expression analysis using a set of HTR-8/SVneo-SP and -NSP cells revealed that SP cells overexpressed several stemness genes including caudal type homeobox2 (CDX2) and bone morphogenic proteins (BMPs), and lymphocyte antigen 6 complex locus D (LY6D) gene was the most highly up-regulated in HTR-8/SVneo-SP cells. LY6D gene reduced its expression in the course of a 7-day cultivation in differentiation medium. SP cells tended to reduce its fraction by treatment of LY6D siRNA indicating that LY6D had potential to maintain cell proliferation of HTR-8/SVneo-SP cells. On ontology analysis, epithelial-mesenchymal transition (EMT) pathway was involved in the up-regulated genes on microarray analysis. HTR-SVneo-SP cells showed enhanced migration. This is the first report that LY6D was important for the maintenance of HTR-8/SVneo-SP cells. EMT was associated with the phenotype of these SP cells..
||Satoru Kyo, Kiyoko Kato, Endometrial Cancer Stem Cell as a Potential Therapeutic Target., Semin Reprod Med, 33, 5, 341-349, 2015.09, Adult stem cells have recently been identified in several types of mature tissue and it has been also suggested that stem-like cells exist in cancerous tissues. It is believed that many cancer stem cells (CSCs) upregulate the expression of drug transporters, allowing them to efficiently pump antitumor agents out of the cells. CSCs reside in a quiescent state, making them resistant to chemotherapeutic agents that target rapidly cycling cells. They are also endowed with a more invasive and metastatic phenotype. These results indicate the requirement to develop a new target treatment for CSCs. There are several methods for the identification of CSCs; for example, detection by CSC markers, such as CD133, CD44, CD117(c-kit), aldehyde dehydrogenase 1 (ALDH1), and isolation of side population (SP), which are identified based on their ability to remove intracellular Hoechst 33342, a fluorescent dye. Here, we review recent articles that show the presence of stem cells in endometrial cancer and introduce the results of our own recent studies using CD133 or CD117 positive cells and SP cells..
||Kanako Okamoto, Ryosuke TSUNEMATSU, Tomoko Tahira, Kenzo Sonoda, KAZUO ASANOMA, Hiroshi Yagi, Tomoko Yoneda, Kenshi Hayashi, Norio Wake, Kiyoko Kato, SNP55, a new functional polymorphism of MDM2-P2 promoter, contributes to allele-specific expression of MDM2 in endometrial cancers, BMC MEDICAL GENETICS, 10.1186/s12881-015-0216-8, 16, 1, 67, 2015.08, BACKGROUND:
The functional single nucleotide polymorphism (SNP) in the MDM2 promoter region, SNP309, is known to be associated with various diseases, particularly cancer. Although many studies have been performed to demonstrate the mechanism of allele-specific expression (ASE) on SNP309, they have only utilized in vitro techniques. It is unknown whether ASE of MDM2 is ascribed solely to SNP309, in vivo.
We attempted to evaluate ASE of MDM2 in vivo using post-labeling followed by automated capillary electrophoresis under single-strand conformation polymorphism conditions. For measuring a quantitative difference, we utilized the SNPs on the exons of MDM2 as markers, the status of which was heterozygous in a large population. To address the cause of ASE beyond 20 %, we confirmed sequences of both MDM2-3'UTR and promoter regions. We assessed the SNP which might be the cause of ASE using biomolecular interaction analysis and luciferase assay.
ASE beyond 20 % was detected in endometrial cancers, but not in cancer-free endometria samples only when an SNP rs1690916 was used as a marker. We suspected that this ASE in endometrial cancer was caused by the sequence heterogeneity in the MDM2-P2 promoter, and found a new functional polymorphism, which we labelled SNP55. There was no difference between cancer-free endometria and endometrial cancer samples neither for SNP55 genotype frequencies nor allele frequencies, and so, SNP55 alone does not affect endometrial cancer risk. The SNP55 status affected the DNA binding affinity of transcription factor Sp1 and nuclear factor kappa-B (NFκB). Transcriptional activity of the P2 promoter containing SNP55C was suppressed by NFκB p50 homodimers, but that of SNP55T was not. Only ASE-positive endometrial cancer samples displayed nuclear localization of NFκB p50.
Our findings suggest that both the SNP55 status and the NFκB p50 activity are important in the transcriptional regulation of MDM2 in endometrial cancers..
||Hitomi Okabe, Shintaro Makino, Kiyoko Kato, Kikumi Matsuoka, Hioyuki Seki, Satoru Takeda, The effect of progesterone on genes involved in preterm labor. , J Reprod Immunol, 10.1016/j.jri.2014.03.008, 104-105, 80-91, 2014.10, The decidua is known to be a major source of intrauterine PGF2α during late gestation and labor, and inflammatory cytokines, including IL-1β, IL-6, and IL-8, are elevated in spontaneous preterm deliveries. In the present study, to elucidate how progesterone blocks the pathways associated with preterm birth, we determined the effects of P4 on the expression of PTGS-2 and PTGFR mRNA in human decidua fibroblast cells, as well as the genes, using microarray analysis. Senescence was induced in primary cultured human decidual cells treated with IL-1β. The IL-1β treatment implicated by microarray analysis increased gene expression levels of PTGS-2, PTGFR, NFκ-B p65, IL-17, and IL-8. In contrast, P4+IL-1β decreased the expression levels of all of these genes in comparison to treatment with IL-1β alone (p<0.05). IL-1β also increased the proportion of SA-β-gal-positive cells. Treatment with IL-1β also increased the p21 protein level in comparison to cells treated either with the vehicle or P4. Neither the p21 protein level nor the number of SA-β-gal-positive cells was increased in normal endometrial glandular cells by IL-1β (p<0.05). Our studies demonstrated that P4 changes the level of gene expression in a manner that favors an anti-inflammatory milieu. Because IL-8 appears to be the cytokine whose expression is most significantly modulated by P4, further studies evaluating IL-8 as a therapeutic target are needed. .
||Nurismangul Yusuf, Tetsunori Inagaki, Soshi Kusunoki, Hitomi Okabe, Izumi Yamada, Akemi Matsumoto, Yasuhisa Terao, Satoru Takeda, Kiyoko Kato, SPARC was overexpressed in human endometrial cancer stem-like cells and promoted migration activity.
, Gynecologic Oncology, 134, 2, 356-363, 2014.08, Objectives
We previously demonstrated that side-population (SP) cells found in human endometrial cancer tissue have features of cancer stem cells (CSCs). Endometrial cancer SP cells show enhanced migration, the potential to differentiate into the mesenchymal cell lineage, and they are associated with the epithelial–mesenchymal transition (EMT). In this study, we analyzed the expression and function of a specific protein, SPARC (secreted protein acidic and rich in cysteine) which we found to be up-regulated in endometrial cancer.
We performed microarray expression analysis to screen for up-regulated genes in CSCs using a set of RK12V-SP cells and -non-SP (NSP) cells. We used the MetaCore package to identify the Gene GO pathway MAPs associated with the up-regulated genes. Here, we investigated the expression and functions of SPARC, one of the genes up-regulated in endometrial CSCs.
We established SPARC-overexpressing cells by transfecting endometrial cancer cells (Ishikawa cells [IK-SPARC cells]). We characterized these cells' growth rate, tumorigenicity, migration and invasion activity. The levels and locations of SPARC protein expression in Hec1SP cells-derived tumors and endometrial cancer tissues were examined by immunohistochemistry.
SPARC was detected by microarray expression analysis during screens for up-regulated genes in SP and NSP CSC. The level of SPARC expression was enhanced in Hec1 SP cells compared with that in Hec1 non-SP cells. SPARC enhanced fibronectin expression and promoted migration activity in IK cells. SPARC expression suppressed tumor growth but promoted formation of tumor stroma.
SPARC was expressed in endometrial cancer tissues, in particular, poorly differentiated endometrioid adenocarcinoma, clear and serous adenocarcinoma,but not in normal endometrial tissue.
This is the first report of overexpression of SPARC in endometrial cancer stem-like cells. SPARC expression is associated with cell migration and stroma formation.
SPARC; Endometrial cancer; EMT; CSCs; Cell migration
||Soshi Kusunoki, Kiyoko Kato, Kouichi Tabu, Tetsunori Inagaki, Hitomi Okabe, Hiroshi Kaneda, Shin Suga, Yasuhisa Terao, Tetsuya Taga, Satoru Takeda, The inhibitory effect of salinomycin on the proliferation, migration and invasion of human endometrial cancer stem-like cells, GYNECOLOGIC ONCOLOGY, 10.1016/j.ygyno.2013.03.005, 129, 3, 598-605, 2013.06, Goals: We previously demonstrated that side-population (SP) cells in human endometrial cancer cells (Hec1 cells) and in rat endometrial cells expressing oncogenic human K-Ras protein (RK12V cells) have features of cancer stem cells (CSCs). Hec1-SP cells showed enhanced migration and
the potential to differentiate into the mesenchymal cell lineage. In this study, we analyzed the association of the epithelial-mesenchymal transition (EMT) with the properties of these endometrial CSCs. We also assessed and the effects of salinomycin (a compound with EMT-specific toxicity) on the proliferative capacity, migration and invasiveness of these endometrial CSCs using Hec1-SP cells.
Method: We performed microarray expression analysis to screen for up-regulated genes in CSCs using a set of RK12V-SP cells and –non-SP(NSP) cells and used the Metacore package to identify the Gene GO pathway MAPs involved in the up-regulated genes. To analyze their association with EMT, the expression of several EMT associated genes in Hec1-SP cells was investigated by real time PCR and compared with that in Hec1-NSP cells. We assessed the expression of BAX, BCL2, LEF1, cyclinD and fibronectin by real time PCR. We also evaluated the viabilities, migration and invasive activities, and tumorigenicities of these SP cells and NSP cells in the presence or absence of salinomycin.
Results: We demonstrated that i) EMT processes were observed in both RK12V-SP cells and Hec1-SP cells, ii) the level of fibronectin was enhanced in Hec1-SP cells and salinomycin reduced the level of fibronectin expression, iii) salinomycin induced apoptosis and inhibited Wnt signaling, and iv) salinomycin inhibited the proliferation, migration, invasiveness and tumorigenicity of these SP cells.
Conclusion: This is the first report of an inhibitory effect of salinomycin on the properties of endometrial CSCs..
||Tomoko Yoneda, Ayumi Kuboyama, Kiyoko Kato, Tatsuhiro Ogami, Kanako Okamoto, Toshiaki Saito, Norio Wake, Association of MDM2 SNP309 and TP53 Arg72Pro polymorphisms with risk of endometrial cancer, ONCOLOGY REPORTS, 10.3892/or.2013.2433, 30, 1, 25-34, 2013.07.
||Kato K, Kuhara A, Yoneda T, Inoue T, Takao T, Ohgami T, Dan L, Kuboyama A, Kusunoki S, Takeda S, Wake N: , Sodium Butyrate inhibitis the Self-Renewal Capacity of Endometrial Tumor Side-Population Cells by Induction a DNA Damage Response.
, Mol Cancer Ther.
, 10, 8, 1-10, 2011.09.
||Kato K, Takao T, Kuboyama A, Tanaka Y, Ohgami T, Yamaguchi S, Adachi S, Yoneda T, Ueoka Y, Kato K, Hayashi S, Asanoma K, Wake N: , Endometrial cancer side-population cells show prominent migration and have a potential to differentiate into the mesenchymal cell lineage. , Am J Pathol., 176, 1, 381-392, 2010.01.
||Inoue T, Kato K, Kato H, Asanoma K, Kuboyama A, Ueoka Y, Yamaguchi S, Ohgami T, Wake N, Level of reactive oxygen species induced by p21Waf1/CIP1 is critical for the determination of cell fate. , Cancer Sci., 100, 7, 1275-1283, 2009.07.
||Tanaka Y, Wake N, Kato K, Letter to Editor, Menopause, in press, 2009.05.
||Inoue T, Kato K, Kato H, Asanoma K, Kuboyama A, Oogami T, Ueoka Y, Wake N, The level of reactive oxygen species induced by p21 WAF1/CIP1 is critical the determination of cell
, Cancer Sci, in press, 2009.05.
||Tanaka Y , Kato K , Mibu R , Uchida S , Asanoma K , Hashimoto K , Nozaki M , Wake N, Medroxyprogesterone acetate inhibits proliferation of colon cancer cell lines by modulating cell
cycle-related protein expression
, Menopause, 15:442-453, 2008.05.
||Kato K, Yoshimoto M, Kato K, Adachi S, Yamayoshi A, Arima T, Asanoma K, Kyo S, Nakahata T, Wake N, Characterization of side population cells (SP cells) in human normal endometrial cells
, Human Reproduction, 22:1214-23, 2007.04.
||Ninomiya Y, Kato K, Takahashi A, Ueoka K, Kamikihara T, Arima T, Matsuda T,Kato H, Nishida J, Wake N,, K-Ras and H-Ras activation promote distinct consequences on endometrial cell survival., Cancer Research, 10.1158/0008-5472.CAN-3487-2, 64, 8, 2759-2765, 64, 2759-2765, 2004.04.