||Yasunaga M¹, Yahata H², Okugawa K¹, Hori E¹, Kodama K¹, Yagi H¹, Ohgami T¹, Onoyama I¹, Asanoma K¹, Kato K¹, Prognostic impact of the subclassification of Müllerian cancer stage IV in the FIGO 2014 staging system with a focus of extra-abdominal lymph node metastases, Int J Clin Oncol, 10.1007/s10147-021-01908-w, 26, 7, 1330-1335, 2021.07, Background: The International Federation of Gynecology and Obstetrics (FIGO) staging system for Müllerian cancer was changed in 2014. Our objective was to evaluate the prognostic impact of stage IV subclassification in this new staging system, especially focusing on extra-abdominal lymph node metastasis.
Methods: Eighty-two patients with stage IV Müllerian cancer treated between 2005 and 2016 at our hospital were retrospectively analyzed. Data for the following clinicopathological variables were analyzed: (1) FIGO stage; (2) tumor stage; (3) lymph node status; (4) histologic type; (5) neoadjuvant chemotherapy; (6) optimal surgery; and (7) bevacizumab use. Survival analysis was performed using Kaplan-Meier curves, log-rank tests, and Cox proportional hazards models.
Results: In accordance with the new classification, 28 and 54 patients were classified as FIGO IVA and IVB, respectively. In the Cox proportional hazards model, early-stage tumors (T1b-3b) and optimal surgery were statistically significant favorable prognostic factors. However, the new FIGO system did not discriminate prognostically between stage IVA and IVB. Median overall survival of stage IVB patients diagnosed with extra-abdominal lymph node metastasis only was better than that of stage IVA and stage IVB patients diagnosed with solid organ metastasis.
Conclusions: In this analysis of the revised FIGO system of patients reclassified as FIGO stage IVA or IVB, no new prognostic information was obtained. There is a possibility that stage IVB patients diagnosed with extra-abdominal lymph node metastasis only can be classified as an earlier stage. Further modification of the FIGO staging system may be needed to improve the prediction of patient prognosis.
Keywords: Extra-abdominal lymph nodes; Müllerian cancer; Prognosis; Stage IV subclassification..
||Sato Y¹, Hidaka N², Sakai A³, Kido S⁴, Fujita Y⁵, Okugawa K⁶, Yahata H⁷, Kato K⁸, Evaluation of the efficacy of vaginal progesterone in preventing preterm birth after abdominal trachelectomy, Eur J Obstet Gynecol Reprod Biol, 10.1016/j.ejogrb.2021.02.009, 259, 119-124, 2021.04, Objective: To determine whether vaginal progesterone (VP) reduces the rate of preterm birth in pregnant women after abdominal trachelectomy (AT) for early-stage cervical cancer STUDY DESIGN: This is an interventional study with a historical cohort. For the interventional study participants who had singleton pregnancies after AT between October 2016 and September 2020, the administration of vaginal progesterone was started between 16+ and 19+6 weeks of gestation and discontinued at 34 weeks of gestation or at the time of delivery, rupture of membranes, or massive uterine bleeding. We investigated obstetric and neonatal outcomes among the study participants and compared them with outcomes of the historical control group participants, included women with singleton pregnancies after AT who were managed without VP at our institution between January 2007 and September 2016, using Fisher's exact test and the Mann-Whitney U test The main outcomes were the gestational age at delivery and incidence of preterm birth before 37 weeks and 34 weeks of gestation.
Result: Twelve pregnancies in ten women were included in the VP group. In contrast, 19 pregnancies in 17 women were included in the historical control group. The incidence of preterm birth at <37 weeks was 10/12 (83 %) in the VP group and 11/19 (58 %) in the control group. The incidence of preterm birth at <34 weeks was 6/12 (50 %) in the VP group and 9/19 (48 %) in the control group. The incidence of preterm birth in the two groups was similar, and the difference between the two groups was not statistically significant.
Conclusion: The administration of vaginal progesterone did not reduce the rate of preterm birth among pregnant women after AT.
Keywords: Abdominal trachelectomy; Preterm birth; Preterm premature membranes rupture; Vaginal progesterone..
||Kodama K¹, Yahata H², Okugawa K¹, Tomonobe H¹, Yasutake N¹, Yoshida S¹, Yagi H¹, Yasunaga M¹, Ohgami T¹, Onoyama I¹, Asanoma K¹, Hori E¹, Shimokawa M³, Kato K¹, Prognostic outcomes and risk factors for recurrence after laser vaporization for cervical intraepithelial neoplasia: a single-center retrospective study, Int J Clin Oncol, 10.1007/s10147-020-01848-x, 26, 4, 770-776, 2021.04, Background: Cervical intraepithelial neoplasia (CIN) is a precancerous lesion that may progress to invasive cervical cancer without intervention. We aim to examine the prognostic outcomes and risk factors for recurrence after laser vaporization for CIN 3, CIN 2 with high-risk human papillomavirus (HPV) infection, and CIN 1 persisting for more than 2 years.
Methods: Between 2008 and 2016, a total of 1070 patients underwent cervical laser vaporization using a carbon dioxide laser. We performed a retrospective review of their medical records to assess their clinical characteristics, pathologic factors, and prognostic outcomes.
Results: The mean patient age was 34 years (range 18-64 years). The preoperative diagnosis was CIN 1 in 27 patients, CIN 2 in 485 patients, and CIN 3 in 558 patients. Over a median follow-up period of 15 months, the 2-year recurrence rate was 18.9%, and the 5-year recurrence rate was 46.5%. The 2-year retreatment rate was 12.6%, and the 5-year retreatment rate was 30.5%. We diagnosed 9 patients with invasive cancer after treatment; all patients underwent combined multidisciplinary treatment, and there were no deaths during follow-up. The recurrence-free interval was correlated with patient age (hazard ratio [HR], 1.028; 95% CI 1.005-1.051; P = 0.0167), body mass index (HR, 1.052; 95% CI 1.008-1.098; P = 0.0191), and glandular involvement (HR, 1.962; 95% CI 1.353-2.846; P = 0.0004).
Conclusions: Cervical laser vaporization is effective and useful for patients with CIN who wish to preserve fertility. However, patients with glandular involvement, older age, and higher body weight require close follow-up for recurrence.
Keywords: Cervical intraepithelial neoplasia; Laser vaporization; Recurrence-free interval..
||Suzuki I¹ ²,Yoshida S², Tabu K³, Kusunoki¹S¹, Matsumura Y², Izumi H⁴, Asanoma K², Yagi H², Onoyama I², Sonoda K⁵, Kohno K⁶, Taga T³, Itakura A¹, Takeda S¹, Kato K⁷, YBX2 and cancer testis antigen 45 contribute to stemness, chemoresistance and a high degree of malignancy in human endometrial cancer, Sci Rep, 10.1038/s41598-021-83200-5, 11, 1, 4220-4220, 2021.02, Y-box binding protein 2 (YBX2) has been associated with the properties of both germ cells and cancer cells. We hypothesized that YBX2 might contribute to the characteristics of cancer stem cells (CSCs). In this study, we clarified the function of YBX2 in endometrial cancer stem cells. We established a human YBX2-expressing Ishikawa (IK) cell line (IK-YBX2 cells). We analyzed gene expression associated with stemness and isolated SP cells from IK-YBX2 cells. The SP population of IK-YBX2 cells, the expression of ALDH1 and serial sphere-forming capacity were associated with levels of YBX2 expression. IK-YBX2 cells were resistant to anti-cancer drugs. In gene expression analysis, a gene for cancer testis antigen, CT45, was generally overexpressed in IK-YBX2 cells. YBX2-mediated CT45 expression was associated with increased levels of self-renewal capacity and paclitaxel resistance. The level of CT45 expression was enhanced in high-grade and/or advanced stages of human endometrial cancer tissues. We conclude that expression of YBX2 is essential for the stem cell-like phenotype. CT45 contributes to stemness associated with YBX2 and might be related to the progression of endometrial cancer..
||Kawamura T¹, Tomari H¹, Onoyama I¹, Araki H², Yasunaga M¹, Lin C¹, Kawamura K¹, Yokota N¹, Yoshida S¹, Yagi H¹, Asanoma K¹, Sonoda K¹, Egashira K¹, Ito T², Kato K¹, Identification of genes associated with endometrial cell ageing, Mol Hum Reprod, 10.1093/molehr/gaaa078, 27, 2, gaaa078-gaaa078, 2021.02, Ageing of the uterine endometrium is a critical factor that affects reproductive success, but the mechanisms associated with uterine ageing are unclear. In this study, we conducted a qualitative examination of age-related changes in endometrial tissues and identified candidate genes as markers for uterine ageing. Gene expression patterns were assessed by two RNA-sequencing experiments using uterine tissues from wild type (WT) C57BL/6 mice. Gene expression data obtained by RNA-sequencing were validated by real-time PCR. Genes expressing the pro-inflammatory cytokines Il17rb and chemokines Cxcl12 and Cxcl14 showed differential expression between aged WT mice and a group of mice composed of 5- and 8-week-old WT (young) animals. Protein expression levels of the above-mentioned genes and of IL8, which functions downstream of IL17RB, were analysed by quantitative immunohistochemistry of unaffected human endometrium tissue samples from patients in their 20s and 40s (10 cases each). In the secretory phase samples, 3,3'- diaminobenzidine staining intensities of IL17RB, CXCL12 and CXCL14 for patients in their 40s were significantly higher than that for patients in their 20s, as detected by a Mann-hitney U test. These results suggest that these genes are candidate markers for endometrial ageing and for prediction of age-related infertility, although confirmation of these findings is needed in larger studies involving fertile and infertile women.
Keywords: CXCL12; CXCL14; IL17RB; endometrial cell ageing; infertility; quantitative immunohistochemistry..
||Yoshida S¹, Asanoma K¹, Yagi H¹, Onoyama I¹, Hori E¹, Matsumura Y¹, Okugawa K¹, Yahata H¹, Kato K¹, Fibronectin mediates activation of stromal fibroblasts by SPARC in endometrial cancer cells, BMC Cancer, 10.1186/s12885-021-07875-9, 21, 1, 156-156, 2021.02, Background: Matricellular glycoprotein, SPARC is a secreted molecule, that mediates the interaction between cells and extracellular matrix. SPARC functions as a regulator of matrix organization and modulates cell behavior. In various kinds of cancer, strong SPARC expression was observed in stromal tissues as well as in cancer epithelial cells. The function of SPARC in cancer cells is somewhat controversial and its impact on peritumoral stromal cells remains to be resolved.
Methods: We investigated the effects of SPARC expression in endometrial cancer cells on the surrounding stromal fibroblasts using in vitro co-culture system. Changes in characteristics of fibroblasts were examined by analysis of fibroblast-specific markers and in vitro contraction assay.
Results: SPARC induced AKT phosphorylation and epithelial-to-mesenchymal transition, consistent with previous reports. Cancer-associated fibroblasts of endometrial cancer expressed higher levels of mesenchymal- and fibroblast-associated factors and had a stronger contraction ability. Unexpectedly, cancer-associated fibroblasts expressed comparable levels of SPARC compared with fibroblasts from normal endometrium. However, co-culture of normal fibroblasts with SPARC-expressing Ishikawa cells resulted in activation of the fibroblasts. Immunodepletion of SPARC did not affect the activation of fibroblasts.
Conclusions: Our data indicated that SPARC activated fibroblasts only in the presence of fibronectin, which was abundantly secreted from SPARC-expressing endometrial cancer cells. These results suggested that a SPARC-fibronectin-mediated activation of fibroblasts might be involved in enhanced mobility and invasion of cancer cells..
||Kato K¹, Terauchi M², Annual report of the Women's Health Care Committee, Japan Society of Obstet-rics and Gynecology, 2020, J Obstet Gynaecol Res, 10.1111/jog.14417, 47, 1, 52-62, 2021.01, Our committee was founded in 2010 with the goal of improving women's health. This academic year, we focused on five activities: cooperation between the departments of pediatric surgery and obstetrics and gynecology for the treatment of persistent cloaca and Mayer-Rokitansky-Küster-Hauser syndrome in Japan; assessment of the educational training of women's health-care advisers; increasing screening for breast and cervical cancer; conducting the Nationwide Survey of Prescribing Practices for the Treatment of Menopausal Symptoms; and preventing osteoporosis in survivors of gynecologic cancer. The activities of each subcommittee are detailed below. This report is based on the Japanese version of the annual report (Acta Obst Gynaec Jpn 2020;72(6):697-707)..
||Yahata H, Sonoda K, Inoue S, Yasutake N, Kodama K, Yagi H, Yasunaga M, Ohgami T, Onoyama I, Kaneki E, Okugawa K, Asanoma K, Kato K, Is Adjuvant Therapy Necessary for Patients with Intermediate-Risk Cervical Cancer after Open Radical Hysterectomy?, Oncology, 10.1159/000508569, 98, 12, 853-858, 2020.11, Introduction: Adjuvant therapy is usually recommended for patients with intermediate-risk cervical cancer (deep stromal invasion [DSI], lymphovascular space invasion [LVSI], and bulky tumor) after radical hysterectomy. However, we previously reported that DSI, LVSI, and bulky squamous cell carcinoma (SCC) were not correlated with prognosis in multivariate analysis; therefore, the indications we use for adjuvant therapy include complete stromal invasion, not DSI or LVSI or bulky SCC. The objective of this study was to evaluate the adequacy of our therapeutic strategy for cervical cancer after radical hysterectomy.
Methods: We performed 321 type III open radical hysterectomies for cervical cancer between 2001 and 2013. Eighty-two patients with DSI, LVSI, or bulky SCC did not receive adjuvant therapy after radical hysterectomy under informed consent. We retrospectively evaluated the prognosis of these 82 patients.
Results: Forty-two patients had >2/3 DSI and 35 patients had 1/3-2/3 DSI. Five patients had LVSI alone. The mean patient age was 43 years (range, 27-72). Six patients (7%) experienced recurrence during a median follow-up period of 84 months (range, 1-206). Two of the 6 patients with recurrence suffered cervical cancer-related deaths, but the remaining 4 cases are alive without evidence of disease after treatment during a follow-up period of 87-165 months. The 5-year disease-free survival rate was 92.6%, and the 5-year overall survival rate was 96.3%.
Conclusions: Adjuvant therapy for DSI, LVSI, or bulky SCC after open radical hysterectomy might not be necessary. Further data collection is warranted to determine the standard of care for patients with intermediate-risk cervical -cancer..
||Kato M¹ ², Onoyama I¹, Kawakami M¹, Yoshida S¹, Kawamura K¹, Kodama K¹, Hori E¹, Cui L¹, Matsumura Y¹, Yagi H¹, Asanoma K¹, Yahata H¹, Itakura A², Takeda S², Kato K¹, Downregulation of 5-hydroxymethylcytosine is associated with the progression of cervical intraepithelial neoplasia, PLoS One, 10.1371/journal.pone.0241482, 15, 11, e0241482-e0241482, 2020.10, Around the world, cervical cancer is one of the most common neoplastic diseases among women, and the prognosis of patients in an advanced stage remains poor. To reduce the mortality rate of cervical cancer, early diagnosis and treatment are essential. DNA methylation is an important aspect of gene regulation, and aberrant DNA methylation contributes to carcinogenesis and cancer progression in various cancers. Although 5-methylcytosine (5mC) has been analyzed intensively, the function of 5-hydroxymethylcytosine (5hmC) has not been clarified. The purpose of our study was to identify the molecular biomarkers for early diagnosis of cervical tumors due to epigenetic alterations. To assess the clinical relevance of DNA methylation, we used immunohistochemistry (IHC) to characterize the level of 5hmC in 102 archived human cervical intraepithelial neoplasia (CIN) samples and cervical cancer specimens. The level of 5hmC was significantly decreased between CIN2 and CIN3. The progression of cervical tumors is caused by a reduction of TP53 and RB1 because of HPV infection. We observed that Tp53 and Rb1 were knocked down in mouse embryonic fibroblasts (MEF), a model of normal cells. The level of 5hmC was reduced in Tp53-knockdown cells, and the expression levels of DNA methyltransferase 1 (DNMT1) and ten-eleven translocation methylcytosine dioxygenase 1 (TET1) were induced. In contrast, there was no significant change in Rb1-knockdown cells. Mechanistically, we focused on apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) 3B (A3B) as a cause of 5hmC reduction after TP53 knockdown. In the human cell line HHUA with a wild-type TP53 gene, A3B was induced in TP53-knockdown cells, and A3B knockdown recovered 5hmC levels in TP53-knockdown cells. These data indicate that TP53 suppression leads to 5hmC reduction in part through A3B induction. Moreover, IHC showed that expression levels of A3B in CIN3 were significantly higher than those in both normal epithelium and in CIN2. In conclusion, 5hmC levels are decreased between CIN2 and CIN3 through the TP53-A3B pathway. Since A3B could impair genome stability, 5hmC loss might increase the chances of accumulating mutations and of progressing from CIN3 to cervical cancer. Thus, these epigenetic changes could predict whether CINs are progressing to cancer or disappearing..
||Kato M¹ ², Onoyama I¹, Yoshida S¹, Cui L¹, Kawamura K¹, Kodama K¹, Hori E¹, Matsumura Y¹, Yagi H¹, Asanoma K¹, Yahata H¹, Itakura A², Takeda S², Kato K¹, Dual-specificity Phosphatase 6 Plays a Critical Role in the Maintenance of a Can-cer Stem-Like Cell Phenotype in Human Endometrial Cancer, Int J Cancer, 10.1002/ijc.32965, 147, 7, 1987-1999, 2020.10, The prognosis of patients with high-grade or advanced-stage endometrial cancer remains poor. As cancer stem-like cells (CSCs) are thought to be associated with endometrial cancers, it is essential to investigate the molecular mechanisms that regulate endometrial CSCs. Dual-specificity phosphatase 6 (DUSP6) functions as a negative-feedback regulator of MAPK-ERK1/2 signaling, but its role in endometrial cancer remains unknown. We investigated whether DUSP6 is involved in cancer cell stemness using endometrial cancer cell lines and specimens from endometrial cancer patients. DUSP6 induced the expression of CSC-related genes including ALDH1, Nanog, SOX2 and Oct4A, increased the population of cells in the G0/G1 phase, and promoted sphere formation ability. DUSP6 knockdown resulted in reduced cell invasion and metastasis, whereas DUSP6 overexpression inhibited apoptosis under serum-free conditions. Moreover, DUSP6 decreased phosphorylated ERK1/2 and increased phosphorylated Akt levels, which potentially induces CSC features. In patients with endometrial cancers, DUSP6 expression was determined using immunohistochemistry, and based on the results, the patients were dichotomized into high- and low-DUSP6-expression groups. Progression-free survival and overall survival were significantly shorter in the high-DUSP6-expression group. These results suggest that DUSP6 has potential value as a biomarker of CSCs and as a target of therapies designed to eliminate CSCs in endometrial cancer..
||Matsui T¹, Tamoto R², Iwasa A², Mimura M², Taniguchi S¹, Hasegawa T¹, Sudo T¹, Mizuno H¹, Kikuta J¹, Onoyama I³, Okugawa K³, Shiomi M⁴, Matsuzaki S⁴, Morii E⁵, Kimura T⁴, Kato K³, Kiyota Y², Ishii M⁶, Nonlinear Optics with Near-Infrared Excitation Enable Real-Time Quantitative Diagnosis of Human Cervical Cancers, Cancer Res, 10.1158/0008-5472.CAN-20-0348, 80, 17, 3745-3754, 2020.09, Histopathologic analysis through biopsy has been one of the most useful methods for the assessment of malignant neoplasms. However, some aspects of the analysis such as invasiveness, evaluation range, and turnaround time from biopsy to report could be improved. Here, we report a novel method for visualizing human cervical tissue three-dimensionally, without biopsy, fixation, or staining, and with sufficient quality for histologic diagnosis. Near-infrared excitation and nonlinear optics were employed to visualize unstained human epithelial tissues of the cervix uteri by constructing images with third-harmonic generation (THG) and second-harmonic generation (SHG). THG images enabled evaluation of nuclear morphology in a quantitative manner with six parameters after image analysis using deep learning. It was also possible to quantitatively assess intraepithelial fibrotic changes based on SHG images and another deep learning analysis. Using each analytical procedure alone, normal and cancerous tissue were classified quantitatively with an AUC ≥0.92. Moreover, a combinatory analysis of THG and SHG images with a machine learning algorithm allowed accurate classification of three-dimensional image files of normal tissue, intraepithelial neoplasia, and invasive carcinoma with a weighted kappa coefficient of 0.86. Our method enables real-time noninvasive diagnosis of cervical lesions, thus constituting a potential tool to dramatically change early detection. SIGNIFICANCE: This study proposes a novel method for diagnosing cancer using nonlinear optics, which enables visualization of histologic features of living tissues without the need for any biopsy or staining dye..
||Okugawa K¹, Yahata H¹, Sonoda K¹, Ohgami T¹, Yasunaga M¹, Kaneki E¹, Kato K¹, Safety evaluation of abdominal trachelectomy in patients with cervical tumors ≥2 cm: a single-institution, retrospective analysis, J Gynecol Oncol, 10.3802/jgo.2020.31.e41, 31, 4, e41-e41, 2020.07, Objective: For oncologic safety, vaginal radical trachelectomy is generally performed only in patients with cervical cancers smaller than 2 cm. However, because inclusion criteria for abdominal trachelectomy are controversial, we evaluated the safety of abdominal trachelectomy for cervical cancers ≥2 cm.
Methods: We began performing abdominal trachelectomies at our institution in 2005, primarily for squamous cell carcinoma ≤3 cm or adenocarcinoma/adenosquamous carcinoma ≤2 cm. If a positive sentinel lymph node or cervical margin was diagnosed intraoperatively by frozen section, the trachelectomy was converted to a hysterectomy. Medical records of these patients were reviewed retrospectively. Patients who had undergone simple abdominal trachelectomy were excluded from this study.
Results: We attempted trachelectomy in 212 patients. Among the 135 patients with tumors <2 cm, trachelectomy was successful in 120, one of whom developed recurrence and none of whom died of their disease. Among 77 patients with tumors ≥2 cm, trachelectomy was successful in 62, 2 of whom developed recurrence and 1 of whom died of her disease. The overall relapse rate after trachelectomy was 1.6% (0.8% in <2 cm group and 3.2% in ≥2 cm group), and the mortality rate was 0.5% (0% in <2 cm group and 1.6% in ≥2 cm group). Recurrence-free survival (p=0.303) and overall survival (p=0.193) did not differ significantly between the <2 cm and ≥2 cm groups.
Conclusions: Abdominal trachelectomy with intraoperative frozen sections of sentinel lymph nodes and cervical margins is oncologically safe, even in patients with tumors ≥2 cm..
||Tomari H¹ ², Kawamura T¹, Asanoma K¹, Egashira K¹, Kawamura K¹, Honjo K², Nagata Y², Kato K¹, Contribution of Senescence in Human Endometrial Stromal Cells During Prolifer-ative Phase to Embryo Receptivity, Biol Reprod, 10.1093/biolre/ioaa044, 103, 1, 104-113, 2020.06, Successful assisted reproductive technology pregnancy depends on the viability of embryos and endometrial receptivity. However, the literature has neglected effects of the endometrial environment during the proliferative phase on implantation success or failure. Human endometrial stromal cells (hESCs) were isolated from endometrial tissues sampled at oocyte retrieval during the proliferative phase from women undergoing infertility treatment. Primary hESC cultures were used to investigate the relationship between stemness and senescence induction in this population and embryo receptivity. Patients were classified as receptive or non-receptive based on their pregnancy diagnosis after embryo transfer. Biomarkers of cellular senescence and somatic stem cells were compared between each sample. hESCs from non-receptive patients exhibited significantly higher (p < 0.01) proportions of senescent cells, mRNA expressions of CDKN2A and CDKN1A transcripts (p < 0.01), and expressions of genes encoding the senescence-associated secretory phenotype (p < 0.05). hESCs from receptive patients had significantly higher (p < 0.01) mRNA expressions of ABCG2 and ALDH1A1 transcripts. Our findings suggest that stemness is inversely associated with senescence induction in hESCs, and by extension, that implantation failure in infertility treatment may be attributable to a combination of senescence promotion and disruption of this maintenance function in this population during the proliferative phase of the menstrual cycle. This is a promising step towards potentially improving the embryo receptivity of endometrium. The specific mechanism by which implantation failure is prefigured by a loss of stemness among endometrial stem cells, and cellular senescence induction among hESCs, should be elucidated in detail in the future..
||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, 33, 12, 13683-13694, 2019.12, 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.
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