Stem cell related genes and malignant progression in gestational trophoblastic diseases

Human germ cells and embryonic cells are similar to cancer cells from the view that both types of cell undergo deprogramming to a stem cell state and become potentially immortal and invasive. Cancer cells may thus express genes that are in common with those expressed in early embryonic cells, especially genes associated with deprogramming and resumption of the undifferentiated stem cell state. The development and malignant progression of gestational trophoblastic diseases, including hydatidiform moles and choriocarcinoma, may be associated with adoption of undifferentiated stem cell state by placental trophoblasts, with aberrant expression of the stem cell related genes. Oct4, Sox2, FoxD3, Stat3 and Nanog are important transcription factors of stem cells and c-mos is important in meiosis. The mRNA expression patterns of Oct-4, Sox2, FoxD3, Stat3 and Nanog were investigated using quantitative TaqMan real-time RT-PCR method on fresh frozen tissue of 15 hydatidiform moles and 15 first trimester normal placenta and two choriocarcinoma cell lines, JAR and JEG. The diagnosis in most of these cases has been confirmed by fluorescent microsatellite genotyping after microdissection and also with chromosome in situ hybridization to analyze the ploidy. TaqMan probes and primers were designed specific to these five genes. The real-time RT-PCR was performed on the ABI PRISM 7700 Sequence Detection System. The expression of the genes was normalized with respect to that of GADPH, a housekeeping gene. Immunohistochemical distribution of c-mos and Oct-4 using formalin-fixed, paraffin-embedded tissues was also performed in 45 hydatidiform moles, 17 choriocarcinomas, and five placental site trophoblastic tumors. A reduced RNA expression of Oct-4 (p=0.0002), Sox2 (p=0.27), FoxD3 (p=0.03), Stat3 (p=0.04) and Nanog (p=0.001) were found in hydatidiform moles when compared with normal placenta. Statistical significant difference was reached in four genes. The RNA expression of Stat3 in hydatidiform moles that regressed was higher than those that developed persistent gestational trophoblastic neoplasia requiring chemotherapy (p=0.033). C-mos expression was found predominantly in the syncytiotrophoblasts and villous intermediate trophoblasts but not in cytotrophoblasts. On the other hand, Oct-4 expression was found predominantly in cytotrophoblasts and villous intermediate trophoblasts while the expression was weak or absent in the syncytiotrophoblasts. These stem cell related genes may play a role in the pathogenesis of the gestational trophoblastic diseases and may even be a marker for predicting of clinical progression of hydatidiform moles

Single nucleotide polymorphisms of follicle-stimulating hormone receptor are associated with ovarian cancer susceptibility

Epidemiological studies suggested that ovulation was associated with ovarian carcinogenesis. Follicle-stimulating hormone (FSH) played an important role in follicular development and was recently found to affect growth of ovarian epithelial cells. Single nucleotide polymorphisms (SNPs) Thr307Ala and Asn680Ser were two non-synonymous variations in the coding region of the FSH receptor (FSHR) gene. This hitherto first case-control study investigating the association between these two FSHR SNPs and the risk of ovarian cancer involved 202 histopathologically confirmed ovarian cancer patients and 266 age-matched cancer-free control subjects using restriction fragment length polymorphism assay and direct sequencing. Our results demonstrated that the 307Ala and 680Ser carriers were associated with significantly increased risk of developing serous and mucinous types of ovarian cancers (P < 0.0005, OR = 2.60, 95% CI = 1.56-4.34; and P < 0.0005, OR = 2.89, 95% CI = 1.73-4.84, adjusted for age, respectively) but not endometrioid and clear cell types. The two SNPs were found to be in modest linkage disequilibrium, D′ = 0.804 and 0.701, r 2 = 0.581 and 0.406 for the cancer and control groups, respectively. The major haplotype of 307Ala-680Ser was also associated with higher cancer risk (P = 0.033, OR = 1.39, 95% CI = 1.03-1.88), especially for the serous and mucinous carcinomas (P = 0.001, OR = 1.82, 95% CI = 1.27-2.60). Our results suggested that the two FSHR SNPs might affect the susceptibility of women to specific subtypes of ovarian cancer. Different types of ovarian cancer might adopt distinct carcinogenetic pathways. Such understanding may be important in selecting patients for ovulation induction therapy. © 2006 Oxford University Press.link_to_subscribed_fulltex

Single nucleotide polymorphisms (SNPs) in the coding sequence of follicle stimulating hormone receptor (FSHR) are associated with ovarian cancer susceptibility

Background: Follicle stimulating hormone (FSH) and its receptor (FSHR) are important in ovarian functions and may implicate in ovarian carcinogenesis. Single nucleotide polymorphism (SNP) of the FSHR gene has previously been found to affect FSH efficacy. The objective of this study was to investigate possible association between two SNPs of FSHR gene, Ala307Thr and Ser680Asn, and susceptibility to ovarian cancer. Methods: After histological review, genomic DNA was extracted from non-cancer tissue of 203 ovarian-cancer patients and 267 age-matched control subjects who had surgery for benign gynecological lesions. The genotypes at Ala307Thr and Ser680Asn were assessed by restriction fragment length polymorphism (RFLP) assay. Results: Genotype frequencies were as listed in the table. The genotypes were in Hardy-Weinburg equilibrium for the control group. Homozygous Thr/Thr for SNP Ala307Thr and Asn/Asn for SNP Ser680Asn seem to carry lower risk of ovarian cancer (OR=0.66; 95%CI=0.45-0.96; p=0.030 and OR=0.60; 95%CI=0.41-0.86; p=0.007 respectively). The protective effect was more pronounced for ovarian cancer of serous (OR=0.43; 95%CI=0.24-0.77; p=0.004 for SNP Ala307Thr; OR=0.43; 95%CI=0.24-0.76; p=0.004 for SNP Ser680Asn) and mucinous (OR=0.40; 95%CI=0.18-0.89; p=0.030 for SNPAla307Thr; OR=0.24; 95%CI=0.10-0.56; p<0.001 for SNP Ser680Asn) types. Interestingly, no association was found between these two SNPs with ovarian cancer of endometrioid and clear cell types. Preliminary analysis of FSHR mRNA and protein expression on different histological types of ovarian cancers by reverse transcription (RT)-PCR and immunohistochemistry showed that expression was found in serous and mucinous types of ovarian cancers but not the endometrioid and clear cell carcinomas. Conclusions: Our results suggest that SNPs at these two sites may affect FSHR function and the susceptibility of the women to ovarian cancer. Serous and mucinous ovarian cancer may adopt different carcinogenetic pathways when compared with endometrioid and clear cell carcinomas known to be associated with endometriosis. The possibility of linkage disequilibrium of these two SNPs in Chinese and their effect in combination are being assessed

Tumor suppressor effect of follistatin-like 1 in ovarian and endometrial carcinogenesis - A differential expression and functional analysis

Endometrial and ovarian cancers are the most common and the most lethal gynecologic malignancies worldwide, respectively. By performing differential expression analysis using annealing control primer™-based reverse transcription (RT)-polymerase chain reaction (PCR) on pooled complementary DNA (cDNA) from 45 endometrial and 36 ovarian cancers and their non-tumor samples, reduced expression of the follistatin-like 1 (FSTL1) was identified. Downregulation of FSTL1 was further confirmed on individual samples and cell lines by quantitative real-time RT-PCR and western blotting. For in vitro functional study, full-length cDNA of FSTL1 was cloned and transiently transfected into the ovarian cancer cell line Ovca420 and endometrial cancer cell line AN3CA. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and cell count demonstrated significantly slower proliferation rate. By terminal uridine deoxynucleotidyl transferase dUTP nick end labeling and flow cytometric analysis, higher apoptotic activity and a remarkable increase in sub-G 1 cell population were observed in transfected cells, suggesting that FSTL1 induced apoptosis in cancer cells. Subsequent messenger RNA and protein expression analysis on downstream apoptotic molecules revealed upregulation and/or activation of FAS, FASLG, TRADD, Caspase-3, Caspase-7 and PARP by FSTL1 transfection, suggesting that FSTL1 -induced apoptosis may be initiated mainly by FAS/FASLG death receptor-ligand binding. Cell migration and invasion assays demonstrated a remarkably lower cell migration and invasion capability in FSTL1 -transfected cells in relation to downregulation of matrix metallopeptidase-2. Our findings suggested that a tumor suppressor role of FSTL1 may be important in ovarian and endometrial carcinogenesis. © The Author 2008. Published by Oxford University Press. All rights reserved.link_to_subscribed_fulltex

Differential expression and phosphorylation of Pak1 and Pak2 in ovarian cancer: Effects on prognosis and cell invasion

Ovarian cancer is a gynecological malignancy with high mortality. Therefore, the identification of novel prognostic and therapeutic targets is important. p21-activated kinases (Paks) are involved in cytoskeleton reorganization. This study investigated the clinical significance of total and phosphorylated (p) Pak1 and Pak2 as well as their functional roles in ovarian cancer. Expressions of Pak1, p-Pak1 Thr 212, Pak2 and p-Pak2 Ser 20 in ovarian normal and cancerous cell lines as well as in clinical samples of ovarian tumors were evaluated. The effects of Pak1 and Pak2 on ovarian cancer cell functions were determined. Pak1, p-Pak1 and p-Pak2 were overexpressed in ovarian cancer cell lines, and clinical samples of ovarian cancers were compared with benign ovarian lesions/inclusion cysts. Similar Pak2 expression levels were observed among normal and cancerous cell lines and clinical samples. After multiple testing correction, high Pak1 and nuclear p-Pak1 expression in ovarian cancers was significantly associated with histological type and tumor grade, respectively. Pak1 and p-Pak1 expression was associated with poor overall and disease-free survival. Pak1 was an independent prognostic factor. Knockdown of Pak1 and Pak2 in ovarian cancer cell lines reduced cell migration and invasion but did not affect cell proliferation and apoptosis. Knockdown of Pak1 also reduced p38 activation and downregulated vascular endothelial growth factor. Conversely, ectopic Pak1 overexpression enhanced ovarian cancer cell migration and invasion in a kinase-dependent manner, along with increased p38 activation. Our findings suggest that Pak1, p-Pak1 and p-Pak2 play important roles in ovarian carcinogenesis. Pak1 and p-Pak1 may be potential prognostic markers and therapeutic molecular targets in ovarian cancer. © 2010 UICC.link_to_subscribed_fulltex

Tyrosine kinase B receptor and BDNF expression in ovarian cancers - Effect on cell migration, angiogenesis and clinical outcome

In this report, we demonstrated that overexpression of tropomyosin-related kinase B (TrkB) was associated with shorter survival in ovarian cancer patients. Brain-derived neurotrophic factor (BDNF), the TrkB ligand, induced activation (phosphorylation) of TrkB in a dose dependent manner. Besides demonstrating the effect of BDNF/TrkB pathway in enhancing cancer cell migration and invasion but inhibiting apoptosis, we also report for the first time that exogenous hepatocyte growth factor induced TrkB expression at both mRNA and protein levels as well as phosphorylation. Our findings suggest that BDNF/TrkB pathway is important in ovarian carcinogenesis and TrkB may be a potential therapeutic target for ovarian cancer. © 2009 Elsevier Ireland Ltd. All rights reserved.link_to_subscribed_fulltex

Aberrant activation of hedgehog signaling pathway contributes to endometrial carcinogenesis through Β-catenin

The hedgehog and Wnt signaling pathways play important roles in human cancers with possible interaction. This study aimed at analysis and correlation of the expression of Gli1, a transcriptional factor and target gene of hedgehog signaling pathway, with clinicopathological parameters and expression of Β-catenin, an important member of the Wnt pathway, in normal, hyperplastic and malignant endometrium. Immunohistochemical study on 15 normal endometrium, 14 simple and complex hyperplasia without atypia, 37 atypical complex hyperplasia and 80 endometrial cancers showed significant Gli1 overexpression and Β-catenin nuclear immunoreactivity in endometrial cancers and atypical endometrial hyperplasia when compared with normal endometrium (P0.05). Overexpression of Gli1 in endometrial cancers correlated with well-differentiated histological grade (P0.001), non-myometrial invasion (P0.004) and superficial myometrial invasion (P0.041). Β-Catenin nuclear immunoreactivity was also associated with well-differentiated histology (P0.013). Gli1 overexpression positively correlated with Β-catenin nuclear immunoreactivity in atypical complex hyperplasia (P0.013) and endometrial carcinoma (P0.017). Similar Gli1 and Β-catenin protein expression pattern was observed in normal and endometrial cancer cell lines by western blotting. We further showed a complex formation between Gli1 and Β-catenin protein in endometrial cancer cell lines in an immunoprecipitation study. Ectopic overexpression of Gli1 into endometrial cancer cells led to reduced expression of Β-catenin in cell cytoplasm and increased expression of Β-catenin in the nuclei. In summary, overexpression of Gli1 was an early event in endometrial carcinogenesis. Aberrant activation of hedgehog pathway may play important roles in endometrial cancer through Β-catenin nuclear accumulation. © 2009 USCAP, Inc. All rights reserved.link_to_OA_fulltex

Hypermethylation of RAS effector related genes and DNA methyltransferase 1 expression in endometrial carcinogenesis

Epigenetic aberration is known to be important in human carcinogenesis. Promoter methylation status of RAS effector related genes, RASSF1A, RASSF2A, hDAB2IP (m2a and m2b regions) and BLU, was evaluated in 76 endometrial carcinomas and their non-neoplastic endometrial tissue by methylation specific PCR. Hypermethylation of at least one of the 5 genes was detected in 73.7% of carcinomas. There were significant correlations between methylation of hDAB2IP and RASSF1A, RASSF2A (p = 0.042, p = 0.012, respectively). Significantly, more frequent RASSF1A hypermethylation was found in Type I endometrioid carcinomas than Type II carcinomas (p = 0.049). Among endometrioid cancers, significant association between RASSF1A hypermethylation and advanced stage, as well as between methylation of hDAB2IP at m2a region with deep myometrial invasion (p < 0.05) was observed. mRNA expression of RASSF1, RASSF2A and BLU in endometrial cancer cell lines significantly increased after treatment with the demethylating agent 5-Aza-2′-deoxycytidine supporting the repressive effect of hypermethylation on their transcription. Immunohistochemical study of DNMT1 on eight normal endometrium, 16 hyperplastic endometrium without atypia, 40 atypical complex hyperplasia and 79 endometrial carcinomas showed progressive increase in DNMT1 immunoreactivity from normal endometrium to endometrial hyperplasia and endometrioid carcinomas (p = 0.001). Among carcinomas, distinctly higher DNMT1 expression was observed in Type I endometrioid carcinomas (p < 0.001). DNMT1 immunoreactivity correlated with RASSF1A and RASSF2A methylation (p < 0.05). The data suggested that hypermethylation of RAS related genes, particularly RASSF1A, was involved in endometrial carcinogenesis with possible divergent patterns in different histological types. DNMT1 protein overexpression might contribute to such aberrant DNA hypermethylation of specific tumor suppressor genes in endometrial cancers. © 2008 Wiley-Liss, Inc.link_to_subscribed_fulltex

Tranexamic acid-associated necrosis and intralesional thrombosis of uterine leiomyomas: A clinicopathologic study of 147 cases emphasizing the importance of drug-induced necrosis and early infarcts in leiomyomas

INTRODUCTION: Women with menorrhagia have increased levels of plasminogen activators in the endometrium. Tranexamic acid (cyklokapron), an antifibrinolytic agent, is commonly prescribed worldwide to women with menorrhagia, including those with fibroids. Necrosis in uterine leiomyomas may be associated with pregnancy, and progestogen or oral contraceptive use but its association with tranexamic acid has not been investigated. Four hundred ninety patients with uterine leiomyomas in 2004 and 2005 were reviewed. Their ages ranged from 22 to 86 (mean 47.2). One hundred forty-seven (30%) were treated with tranexamic acid. RESULTS: Infarct-type necrosis was observed in the leiomyomas of 38 patients, 22 of whom had tranexamic acid (15%) whereas the remaining 16 had no drug exposure (4.7%) (odds ratio=3.60; 95% confidence interval: 1.83-6.07; P=0.0003). Two patients who took the drug less than 2 weeks before surgery had early infarcts with appearance resembled coagulative type necrosis. Eleven of the 22 cases of drug-induced necrotic leiomyoma (50%) also showed intralesional thrombus formation, and 4 showed organization of the thrombi. CONCLUSIONS: Infarct-type necrosis and thrombosis of leiomyoma was more commonly observed in patients treated with tranexamic acid. Although the drug is effective for menorrhagia, clinicians should be aware of the possible complications associated with leiomyoma necrosis such as pain and fever. Distinguishing between types of necrosis may not always be straightforward particularly in early infarcts when the reparative connective tissue reaction between the viable and necrotic cells is not well-developed, resulting in an appearance similar to coagulative necrosis. When the overall gross and microscopic features of a leiomyoma with coagulative necrosis favor a benign lesion, the drug history should be reviewed so that this type of early and healing infarct-type necrosis is considered as the underlying cause of the apparent coagulative necrosis. This may otherwise result in a diagnosis of smooth muscle tumor of uncertain malignant potential, leading to prolonged follow-up and unnecessary further surgical intervention. © 2007 Lippincott Williams & Wilkins, Inc.link_to_subscribed_fulltex