Tatsuro Furui

The PTEN/MMAC1/TEP tumor suppressor gene decreases cell growth and induces apoptosis and anoikis in breast cancer cells

The PTEN/MMAC1/TEP (PTEN) tumor suppressor gene at 10q23.3 is mutated in multiple types of sporadic tumors including breast cancers and also in the germline of patients with the Cowdens breast cancer predisposition syndrome. The PTEN gene encodes a multifunctional phosphatase capable of dephosphorylating the same sites in membrane phosphatidylinositols phosphorylated by phosphatidylinositol 3′-kinase (PI3K). We demonstrate herein that loss of PTEN function in breast cancer cells results in an increase in basal levels of phosphorylation of multiple components of the P13K signaling cascade as well as an increase in duration of ligand-induced signaling through the P13K cascade. These alterations are reversed by wild-type but not phosphatase inactive PTEN. In the presence of high concentrations of serum, enforced expression of PTEN induces a predominant G1 arrest consistent with the capacity of PTEN to evoke increases in the expression of the p27Kip1 cyclin dependent kinase inhibitor. In the presence of low concentrations of serum, enforced PTEN expression results in a marked increase in cellular apoptosis, a finding which is consistent with the capacity of PTEN to alter the phosphorylation, and presumably function, of the AKT, BAD, p70S6 kinase and GSK3α apoptosis regulators. Under anchorage-independent conditions, PTEN also induces anoikis, a form of apoptosis that occurs when cells are dissociated from the extracellular matrix, which is enhanced in conjunction with low serum culture conditions. Together, these data suggest that PTEN effects on the PI3K signaling cascade are influenced by the cell stimulatory context, and that depending on the exposure to growth factors and other exogenous stimuli such as integrin ligation, PTEN can induce cell cycle arrest, apoptosis or anoikis in breast cancer cells.

Lysophospholipid Growth Factors in the Initiation, Progression, Metastases, and Management of Ovarian Cancer

Abstract: Levels of lysophosphatidic acid (LPA) and lysophosphatidylcholine (LPC) are elevated in the plasma and ascites of ovarian cancer patients, but not in most other tumor types. LPA increases cell proliferation, cell survival, resistance to cisplatin, cell shrinkage, and production of vascular endothelial growth factor, urokinase plasminogen activator, and LPA itself in ovarian cancer cells, but not in normal ovarian surface epithelial cells. PSP24 and members of the endothelial differentiation gene (EDG) family (EDG1, EDG2, EDG4, and EDG7) of G protein‐coupled receptors mediate LPA signaling. Ovarian cancer cell lines do not express EDG1 mRNA, have variable EDG2 mRNA and protein levels, and frequently exhibit elevated levels of EDG4 mRNA and protein, suggesting that EDG4 may contribute to the deleterious effects of LPA in ovarian cancer. In contrast, activation of the EDG2 LPA receptor on ovarian cancer cells may lead to apoptosis and counter the effects of other LPA receptors. Thus, the development of agonists and antagonists for the appropriate spectrum of LPA receptors may alter proliferation, apoptosis, or response to therapy of ovarian cancer cells. Indeed, over 60% of all current drugs target the G protein‐coupled family of receptors, making the LPA receptor family a “drugable” target. LPC, although not as thoroughly studied, increases cellular proliferation and mediates multiple other functions through unique signaling pathways.

Gonadotropin-Releasing Hormone Receptor in Gynecologic Tumors Frequent Expression in Adenocarcinoma Histologic Types

Background. Gonadotropin‐releasing hormone (GnRH) analogs have been used in the therapy of the endocrine‐dependent cancers. The authors attempted to determine the frequency with which Gn‐RH receptor (Gn‐RHR) is present in gynecological cancers.

Critical Role of Lysophospholipids in the Pathophysiology, Diagnosis, and Management of Ovarian Cancer

Lysophosphatidic acid (LPA), the simplest of all phospholipids, exhibits pleiomorphic functions in multiple cell lineages. The effects of LPA appear to be mediated by binding of LPA to specific members of the endothelial differentiation gene (Edg) family of G protein-coupled receptors (GPCR). Edg 2, Edg4, and Edg7 are high affinity receptors for LPA, and Edg1 may be a low affinity receptor for LPA. PSP24 has been shown to be responsive to LPA in Xenopus oocytes, however, its role in mammalian cells is unclear. The specific biochemical events initiated by the different Edg receptors, as well as the biological outcomes of activation of the individual receptors, are only beginning to be determined. LPA levels are consistently elevated in the plasma and ascites of ovarian cancer patients, but not in most other epithelial tumors, with the exception of cervix and endometrium, suggesting that LPA may be of particular importance in the pathophysiology of ovarian cancer. In support of this concept, ovarian cancer cells constitutively and inducibly produce high levels of LPA and demonstrate markedly different responses to LPA than normal ovarian surface epithelium. Edg4 and Edg7 levels are consistently increased in malignant ovarian epithelial cells contributing to the aberrant response of ovarian cancer cells to LPA. Edg2 may represent a negative regulatory LPA receptor inducing apoptosis in ovarian cancer cells. Thus, increased levels of LPA, altered receptor expression and altered responses to LPA may contribute to the initiation, progression or outcome of ovarian cancer. Over 40% of known drugs target GPCR, making LPA receptors attractive targets for molecular therapeutics. Indeed, using the structure-function relationship of LPA in model systems, we have identified selective Edg2 anatgonists, as well as Edg4 and Edg7 agonists. These lead compounds are being assessed in preclinical model systems. Understanding the mechanisms regulating LPA production, metabolism and function could lead to improved methods for early detection and to new targets for therapy in ovarian cancer.

Evidence for coupling of phosphotyrosine phosphatase to gonadotropin-releasing hormone receptor in ovarian carcinoma membrane

Gonadotropin‐releasing hormone (Gn‐RH) receptor (Gn‐RHR) has been demonstrated in epithelial ovarian carcinoma (Imai et al., Cancer 1994;74:2555‐61). To examine whether Gn‐RHR mediates direct antiproliferative effects, we attempted to determine stimulatory regulation by Gn‐RH of phosphotyrosine phosphatase (PTP) activity in plasma membranes isolated from ovarian carcinoma samples.

Presence of gonadotropin-releasing hormone and its messenger ribonucleic acid in human ovarian epithelial carcinoma

Objective: The purpose of this study was to investigate the expression of gonadotropin-releasing hormone messenger ribonucleic acid and the presence of gonadotropin-releasing hormone in human ovarian carcinoma known to have gonadotropin-releasing hormone binding sites and to be affected by gonadotropin-releasing hormone analog. Study Design: Human ovarian carcinomas surgically removed and human ovarian carcinoma cell lines were examined. Gonadotropin-releasing hormone was determined by a radioimmunoassay and a bioassy. Gonadotropin-releasing hormone messenger ribonucleic acid was determined by reverse transcription polymerase chain reaction using oligonucleotide primers synthesized according to the published human gonadotropin-releasing hormone sequence. Results: Gonadotropin-releasing hormone was shown to be present in extracts of ovarian mucinous cystadenocarcinoma sample (0.8 ± 0.12 pg/mg of protein) and ovarian adenocarcinoma cell line SK-OV3 (0.92 ± 0.17 pg/mg of protein) but not in the normal ovary and placenta. Two of two extract samples from individual cases evoked dose-dependent phosphoinositide breakdown in rat granulosa cells similar to that caused by authentic gonadotropin-releasing hormone. Gonadotropin-releasing hormone messenger ribonucleic acid was detected in two of two mucinous cystadenocarcinoma specimens, one of one serous cystadenocarcinoma, and SK-OV3 cells but not in the dysgerminoma, mucinous cystadenoma, and normal ovary and placenta. Conclusion: The demonstration of gonadotropin-releasing hormone and its messenger ribonucleic acid raises the possibility that gonadotropin-releasing hormone may play an autocrine regulatory role in the growth of ovarian carcinoma.

Direct protection by a gonadotropin-releasing hormone analog from doxorubicin-induced granulosa cell damage.

Background/Aims: Recent clinical applications suggest a beneficial effect of gonadotropin-releasing hormone analog (GnRHa) as a gonadal protector from chemotherapy-induced premature ovarian failure. This study aimed to determine cellular mechanisms involved in the protective action of GnRHa against granulosa cell damage caused by doxorubicin. Methods: Granulosa cells were obtained by ultrasound-guided follicular aspiration from patients undergoing in vitro fertilization, and screened for GnRH receptor expression prior to analyses. The cellular function was assessed by measuring the conversion of exogenously supplied androstenedione to estradiol-β (E2) in response to follicle-stimulating hormone (FSH) (1 µM). Results: Exposing to doxorubicin for 12 h before FSH stimulation caused a concentration-dependent inhibition of the E2 secretion to a minimum level of 20% of control. When the cells were incubated with a GnRHa for 12 h before and during exposure to doxorubicin, granulosa cells produced an equal level of E2 to that of control cells. The protective action of GnRHa was dose-dependent; a half-maximal effect occurred at 10 nM. Preincubation with GnRHa alone had no effect on FSH-induced E2 production. Conclusion: These findings demonstrate that a GnRHa may retard doxorubicin-induced granulosa cell damage, suggesting an additional GnRH activity to protect the gonads during chemotherapy through GnRH receptor-mediated mechanism(s).

Cancer stem-like cells of ovarian clear cell carcinoma are enriched in the ALDH-high population associated with an accelerated scavenging system in reactive oxygen species

OBJECTIVE In ovarian cancer cases, recurrence after chemotherapy is frequently observed, suggesting the involvement of ovarian cancer stem-like cells (CSCs). The chemoresistance of ovarian clear cell carcinomas is particularly strong in comparison to other epithelial ovarian cancer subtypes. We investigated the relationship between a CSC marker, aldehyde dehydrogenase 1 (ALDH1), and clinical prognosis using ovarian clear cell carcinoma tissue samples. Furthermore, we investigated the antioxidant mechanism by which CSCs maintain a lower reactive oxygen species (ROS) level, which provides protection from chemotherapeutic agents. METHODS Immunohistochemical staining was performed to examine the CSC markers (CD133, CD44, ALDH1) using ovarian clear cell carcinoma tissue samples (n=81). Clear cell carcinoma cell lines (KOC-7C, OVTOKO) are separated into the ALDH-high and ALDH-low populations by ALDEFLUOR assay and fluorescence-activated cell sorting (FACS). We compared the intracellular ROS level, mRNA level of the antioxidant enzymes and Nrf2 expression of the two populations. RESULTS High ALDH1 expression levels are related to advanced stage in clear cell carcinoma cases. ALDH1 expression significantly reduced progression free survival. Other markers are not related to clinical stage and prognosis. ALDH-high cells contained a lower ROS level than ALDH-low cells. Antioxidant enzymes were upregulated in ALDH-high cells. ALDH-high cells showed increased expression of Nrf2, a key transcriptional factor of the antioxidant system. CONCLUSIONS ALDH-positive CSCs might have increased Nrf2-induced antioxidant scavengers, which lower ROS level relevant to chemoresistance in ovarian clear cell carcinoma.

Gynecologic tumors and symptoms in childhood and adolescence; 10-years' experience

OBJECTIVES: The advancement of diagnostic imaging evaluations and the earlier occurrence of secondary sexual development prompted us to review our recent experience with genital tract tumors in children. METHODS: We analyzed data for 1938 patients aged less than 18 years who were referred to Gifu University School of Medicine‐affiliated Hospitals for the years 1984 through 1993. RESULTS: Of the patients, 145 underwent surgical treatment. Vaginal tumor was seen in 5 patients; 1 endodermal sinus tumor, 1 sarcoma botryoides and 3 Gartners duct cysts. Two patients with malignant tumor presented only with bloody vaginal discharge; recurrent abdominal pain due to vaginal obstruction was noted in 1 patient with the cyst. Ten had benign tumors in the vulva, presenting with a genital mass. Of 114 ovarian tumors, 3 were diagnosed by antenatal ultrasonographic examinations. Fifty‐five had germ cell tumors, 33 had epithelial tumors, and 18 had stromal tumors. The most common symptom was abdominal pain and approximately one‐third of girls who complained of abdominal pain had an ovarian tumor. Precocious puberty was noted in 4 girls with stromal tumor. Two of the 23 malignant tumors developed in the vagina and the others originated in the ovary. In 19 patients unilateral salpingo‐oophorectomy or local excision was done in an attempt for reproductive organ conservation; 4 cases of advanced stage disease were treated with hysterectomy and/or bilateral salpingo‐oophorectomy. Only 3 of the 23 patients with malignant tumor died within 4 years and others are free from disease. CONCLUSIONS: Genital symptoms, even common, alert us to the posibility of a genital tract tumor. The prompt and precise detection of either benign or malignant tumors in children may lead to cure and preservation of fertility with conservative surgery.

Enhancing effects of estrogens on endometrial carcinogenesis initiated by N-methyl-N-nitrosourea in ICR mice

The present study was undertaken to examine the effects of estrogens, such as estrone (E1), 17β‐estradiol (E2) and estriol (E3), on endometrial Carcinogenesis initiated by N‐methyl‐N‐nitrosourea (MNU) in mice. A total of 120 female ICR mice received MNU solution (1 mg/100 g body wt.) and normal saline at 10 weeks of age into their left and right uterine corpora, respectively. One week later, they were divided into four groups and treated as follows: Group 1 (30 mice) was given 25 ppm E1‐containing diet; Group 2 (30 mice) was fed 5 ppm E2‐containing diet; Group 3 (30 mice) was given 25 ppm E3‐containing diet; and Group 4 (30 mice) was fed the basal diet alone. At the termination of the experiment (Week 30), all surviving animals were autopsied and histopathological examinations revealed that endometrial adenocarcinomas had developed in all groups. The incidence of adenocarcinomas in the MNU‐treated uterine corpus in Group 1 (25 ppm E1‐feeding, 9/23, 39%) was significantly higher than that in Group 4 (basal diet, 3/26, 12%, P<0.05). Also, the incidences of adenocarcinomas in the MNU‐treated uterine corpus in Groups 2 (5 ppm E2‐feeding, 8/24, 33%) and 3 (25 ppm E3‐feeding, 7/26, 28%) were higher than in Group 4, but the difference was not statistically significant. Feeding of diet containing E1, E2 and E3 increased the incidences of the preneoplastic endometrial lesions (atypical, adenomatous or cystic glandular hyperplasia). In the uterine cervix, small numbers of squmous cell carcinomas, dysplasias or hyperplasias were occasionally found in all groups. These results indicate enhancing effects of the above three types of estrogens on the endometrial carcinogenesis induced by MNU in ICR mice.