Detection of circulating tumor cells in cervical cancer using a conditionally replicative adenovirus targeting telomerase-positive cells.

Circulating tumor cells (CTC) are newly discovered biomarkers of cancers. Although many systems detect CTC, a gold standard has not yet been established. We analyzed CTC in uterine cervical cancer patients using an advanced version of conditionally replicative adenovirus targeting telomerase-positive cells, which was enabled to infect coxsackievirus-adenovirus receptor-negative cells and to reduce false-positive signals in myeloid cells. Blood samples from cervical cancer patients were hemolyzed and infected with the virus and then labeled with fluorescent anti-CD45 and anti-pan cytokeratin antibodies. GFP (+)/CD45 (-) cells were isolated and subjected to whole-genome amplification followed by polymerase chain reaction analysis of human papillomavirus (HPV) DNA. CTC were detected in 6 of 23 patients with cervical cancers (26.0%). Expression of CTC did not correlate with the stage of cancer or other clinicopathological factors. In 5 of the 6 CTC-positive cases, the same subtype of HPV DNA as that of the corresponding primary lesion was detected, indicating that the CTC originated from HPV-infected cancer cells. These CTC were all negative for cytokeratins. The CTC detected by our system were genetically confirmed. CTC derived from uterine cervical cancers had lost epithelial characteristics, indicating that epithelial marker-dependent systems do not have the capacity to detect these cells in cervical cancer patients.

FDG-PET-positive ovarian thecoma with GLUT5 expression: Five cases.

Positron emission tomography (PET) with fluorodeoxyglucose F18 (18 F-FDG) is useful for detecting malignancies, but benign lesions occasionally have false-positive 18 F-FDG uptake. Here, we report the cases of five postmenopausal women with solid ovarian tumors suspected to be ovarian cancer on magnetic resonance imaging and 18 F-FDG uptake. Mean age of the five patients was 57 years (range, 53-65 years). Average early standardized uptake value (SUV) of 18 F-FDG was 5.76 (range, 2.2-12.0) and delayed SUV was 6.56 (range, 2.4-13.8). In all five patients, frozen section diagnosis at surgery was thecoma, and bilateral salpingo-oophorectomy was performed. On immunohistochemistry, immunoreactive glucose transporter 5 (GLUT5) expression was detected in thecoma tissues. This case shows that thecoma sometimes has positive 18 F-FDG uptake on positron emission tomography-computed tomography (PET-CT), indicating the need for caution regarding false-positive PET-CT in patients with benign solid ovarian tumor.

Tamoxifen-induced ovarian hyperstimulation during premenopausal hormonal therapy for breast cancer in Japanese women.

PURPOSE: Tamoxifen is an anti-estrogenic drug that is widely used for endocrine-dependent breast cancer as adjuvant hormonal therapy, and its use has been reported to be frequently associated with high levels of serum estradiol. Since the population of premenopausal women receiving tamoxifen therapy is growing in Japan, we retrospectively analyzed the incidence of ovarian hyperstimulation by tamoxifen therapy in Japanese women. METHODS: Eleven patients who received surgical therapy for endocrine-dependent breast cancer and showed high values of serum estradiol during post-operative tamoxifen therapy were recruited in this study and evaluated by examining the serum concentration of follicular stimulating hormone (FSH) and follicular development. RESULTS: The mean age, serum concentrations of estradiol and FSH, and follicular diameter were 41.3 years old, 1015.8 pg/mL, 11.8 mIU/mL, and 3.47 cm, respectively. In 6 cases, multiple follicular development was observed, while the other cases showed single follicular development with a mean serum estradiol level of 848.6 pg/mL and follicular diameter of 4.46 cm. There was no significant difference in age or FSH concentration between the two groups. The mean periods from the start of the single administration of tamoxifen to the initial detection of a high estradiol concentration was 716.5 days. CONCLUSIONS: These findings indicate that tamoxifen could stimulate the ovarian function even after 2-year treatment. Since single and multiple follicular developments with large sizes were observed, dual mechanisms through the inhibition of both negative and positive feedback to the hypothalamic-pituitary-axis can be proposed to explain the adverse effects of tamoxifen on ovarian function.

Dienogest, a synthetic progestin, down-regulates expression of CYP19A1 and inflammatory and neuroangiogenesis factors through progesterone receptor isoforms A and B in endometriotic cells.

Dienogest (DNG) is a selective progesterone receptor (PR) agonist and oral administration of DNG is used for the treatment of endometriosis. DNG is considered to act on PR to down-regulate pathophysiological factors associated with endometriosis. PR exists as two major isoforms, PR-A and PR-B, and their physiological functions are mostly distinct. It was suggested that PR isoform expression patterns are altered in endometriosis, but it is unknown whether the pharmacological effects of DNG are exerted through PR-A, PR-B or both. In the present study, we investigated the pharmacological effects of DNG through these PR isoforms on the expression of CYP19A1 which encodes aromatase and inflammatory and neuroangiogenesis factors associated with the pain and progression of endometriosis. We used immortalized human endometriotic epithelial cell lines that specifically express PR-A or PR-B in a spheroid cell culture system, and treated them with DNG. We evaluated messenger RNA (mRNA) expression of CYP19A1, prostaglandin (PG)E2 synthase (cyclooxygenase (COX)-2 and microsomal PGE2 synthase (mPGES)-1), inflammatory cytokines (interleukin (IL)-6, IL-8, and monocyte chemoattractant protein (MCP)-1) and neuroangiogenesis factors (vascular endothelial growth factor (VEGF) and nerve growth factor (NGF)) using real-time polymerase chain reaction. In addition, PGE2 production was measured by enzyme immunoassay. We found that DNG down-regulated mRNA expression of CYP19A1, COX-2, mPGES-1, IL-6, IL-8, MCP-1, NGF and VEGF, and PGE2 production in human endometriotic epithelial cell lines that specifically express either PR-A or PR-B. These results demonstrate that DNG activates both PR-A and PR-B and down-regulates the expression of pathophysiological factors associated with pain and progression of endometriosis. Our results suggest that DNG exerts therapeutic efficacy against the pain and progression of endometriosis regardless of PR isoform expression patterns.

Concurrent estrogen action was essential for maximal progestin effect in oral contraceptives.

OBJECTIVE: To investigate the impact of estrogen contained in oral contraceptives (OCs) on the action of progestin on ovarian endometrioma epithelial cells. DESIGN: Experimental in vitro study and immunohistochemical analysis. SETTING: University hospital. PATIENT(S): Patients who underwent surgery due to ovarian endometrioma. INTERVENTION(S): Not applicable. MAIN OUTCOME MEASURE(S): Telomerase-immortalized epithelial cells derived from ovarian endometrioma were treated with norethindorone (NET; 80 nmol/L) or levonorgestrel (LNG; 20 nmol/L) with or without 17ß-ethynylestradiol (EE; 0.6 nmol/L) for 96 hours, and the cell growth was monitored. Estrogen receptor (ER) α, progesterone receptor (PR) A, and PRB expressions in clinical samples of ovarian endometrioma epithelial cells were analyzed with the use of immunohistochemistry. RESULT(S): NET or LNG effectively suppressed cell growth, and addition of EE significantly enhanced the growth suppression. This EE-mediated enhancement of cell growth suppression was observed only in cells that expressed ERα and therefore was ERα dependent. Western blot analysis revealed that expression of PRB was significantly induced by the addition of EE. Immunohistochemical analysis confirmed that ERα expression and PRB expression are significantly correlated, indicating that progestin-sensitive cells with PRB expression are predisposed to react with estrogen stimulation. CONCLUSION(S): These findings suggest that EE contained in OCs plays a supportive role in progestin-induced growth inhibition of ovarian endometrioma epithelial cells. In the absence of estrogen priming, concurrent estrogen action was essential for rapid induction of PR to achieve maximal progestin effect.

Molecular characterization of CD133+ cancer stem-like cells in endometrial cancer.

A small subset of cells with CD133 expression is thought to have increased chemoresistance and tumorigenicity, features of cancer stem cells (CSCs); the molecular mechanisms by which these properties arise remain unclear. We characterized CD133+ endometrial cancer cells based on microarray analyses of Ishikawa cells. Of the genes upregulated in CD133+ cells compared with CD133- cells, we noted several key factors involved in the aggressive behavior of cells, including ABCG2 and matrix metalloproteinase (MMP). Flow cytometric analyses identified a side-cell population (SP) with CSC features in Ishikawa cells, and they were found to be more enriched in CD133+ cells than CD133- cells. In particular, CD133+/SP cells exhibited higher proliferative and colony­forming activity than CD133+/non-SP cells. Matrigel invasion assay revealed that CD133+ cells have enhanced invasive capacity with elevated MT1-MMP expression. siRNA­based knockdown of MT1-MMP largely abolished the invasive capacity of CD133+ cells, but not CD133- cells due to low levels of constitutive MT1-MMP1 expression. These findings demonstrate that increased chemoresistance and tumorigenic potential of CD133+ cells are at least partly attributed to an enriched SP fraction as well as increased MMP-1 expression. These results will be of assistance in the establishment of molecular target therapy to CSCs in endometrial cancer.

Imatinib sensitizes endometrial cancer cells to cisplatin by targeting CD117-positive growth-competent cells.

The use of molecular target therapy has not been established for endometrial cancer. The present study investigated the potential therapeutic strategy of targeting CD117-positive cancer cells as a novel molecular target therapy. FACS-sorted CD117(+) cells isolated from endometrial cancer cell lines (Ishikawa or MFE280 cells) exhibited higher proliferative capacity in vitro and colony forming activity on soft agar, and decreased sensitivity to cisplatin, compared to CD117(-) cells. Immunohistochemical analyses with surgical specimens of endometrial cancers showed that high CD117 expression was tightly linked to advanced FIGO stages, myometrial invasion and histological grade, and was significantly associated with poor overall survival and relapse-free survival (Kaplan-Meier analysis; p<0.001, log-rank test). The Cox-regression hazard model identified high CD117 expression to be an independent prognostic factor for survival (p<0.05). In vitro assay confirmed that stem cell factor (SCF), a ligand of CD117, was produced specifically in CD117(+) cells of endometrial cancer, and the colony-forming activity were abrogated by adding anti-SCF antibody, indicating an SCF-dependent growth property. Imatinib was confirmed to selectively target CD117(+) cells in vitro, and synergistically enhanced the anti-tumor effect of low dose cisplatin in vivo, which showed only modest effects when used as a single use. These findings suggest that CD117 can be a marker of aggressive behavior of cells as well as an independent prognostic marker in endometrial cancer. Targeting of the SCF/CD117 axis by imatinib sensitized endometrial cancer cells to cisplatin, proposing a novel therapeutic strategy for this tumor type.

The PRB-dependent FOXO1/IGFBP-1 axis is essential for progestin to inhibit endometrial epithelial growth.

Progestin inhibits the growth of normal and cancerous endometria via the progesterone receptor (PR), but the distinct functions and signalings of PR subtypes have not been fully understood. The aim of the present study was to dissect the key pathways of progestin to inhibit endometrial epithelial growth. Immortalized endometrial epithelial cells (EM-E6/E7/TERT) with stable PRA or PRB expression were established and used for the experiments. In vitro growth inhibition by progestin was mainly observed in EM-E6/E7/TERT cells with PRB rather than those with PRA. RT-PCR assay confirmed that FOXO1, a key gene for progestin action, was up-regulated by progestin in a PRB-dependent manner. cDNA microarray analysis identified IGFBP-1, which contains FOXO1 binding sites on its promoter, to be induced by medroxyprogesterone acetate (MPA) in EM-E6/E7/TERT cells with PRB but not with PRA. siRNA knockdown of FOXO1 disturbed the induction of IGFBP-1 by MPA, while IGFBP-1 knockdown showed no effect on MPA-induced FOXO1 expression, indicating that FOXO1 is an upstream regulator of IGFBP-1. Luciferase reporter assays showed that MPA activated the IGFBP-1 promoter, which was cancelled by FOXO1 knockdown. Chromatin immunoprecipitation assay confirmed the in vivo binding of FOXO1 to the core promoter of IGFBP-1. IGFBP-1 knockdown significantly attenuated the growth inhibitory effects of MPA. The FOXO1/IGFBP-1 axis is essential for PRB-dependent growth inhibition of endometrial epithelial cells, offering a potential therapeutic clue to enhance the progestin effect.

Activation of NF-kappaB is a novel target of KRAS-induced endometrial carcinogenesis.

PURPOSE: Although the KRAS mutation is one of critical genetic alterations in endometrial carcinogenesis, the downstream targets are not known. EXPERIMENTAL DESIGN: In this study, we investigated the molecular targets of KRAS signals, using tumorigenic cells with oncogenic KRAS mutation established from telomerase reverse transcriptase (TERT)-immortalized endometrial epithelial cells. RESULTS: We first confirmed that the RAF-ERK pathway, but not the PI3K-Akt pathway, was activated in KRAS tumorigenic cells. However, the introduction of constitutively active MAP/ERK kinase into immortalized cells to mimic RAF-ERK activation failed to obtain tumorigenic phenotypes, indicating the existence of other carcinogenic pathways triggered by KRAS. Recent evidence suggestive of linkage with KRAS signals prompted us to examine the involvement of NF-κB in endometrial carcinogenesis. We found that the DNA-binding activity of NF-κB was markedly elevated in KRAS tumorigenic cells compared with TERT-immortalized cells. Furthermore, the ability of NF-κB to activate the target gene promoters significantly increased in KRAS tumorigenic cells. Introduction of a mutant IκB that is resistant to degradation and thereby enhances the inhibitory effect on NF-κB largely abrogated the transformed phenotypes of KRAS tumorigenic cells. Thus, oncogenic KRAS signals contributed to the tumorigenic phenotypes of endometrial cells by activating the transcription function of NF-κB. CONCLUSIONS: These findings clearly show that NF-κB activation is a novel target of oncogenic KRAS in endometrial carcinogenesis, implying the potential utility of NF-κB inhibitors for endometrial cancer chemoprevention, especially with KRAS mutation.