Nelson S.S. Siu

Dysregulation of microRNA-204 mediates migration and invasion of endometrial cancer by regulating FOXC1

MicroRNAs (miRNAs) regulate mRNA stability and protein expression, and certain miRNAs have been demonstrated to act either as oncogenes or tumor suppressors. Differential miRNA expression signatures have been documented in many human cancers but the role of miRNAs in endometrioid endometrial cancer (EEC) remains poorly understood. This study identifies significantly dysregulated miRNAs of EEC cells, and characterizes their impact on the malignant phenotype. We studied the expression of 365 human miRNAs using Taqman low density arrays in EECs and normal endometriums. Candidate differentially expressed miRNAs were validated by quantitative real‐time PCR. Expression of highly dysregulated miRNAs was examined in vitro through the effect of anti‐/pre‐miRNA transfection on the malignant phenotype. We identified 16 significantly dysregulated miRNAs in EEC and 7 of these are novel findings with respect to EEC. Antagonizing the function of miR‐7, miR‐194 and miR‐449b, or overexpressing miR‐204, repressed migration, invasion and extracellular matrix‐adhesion in HEC1A endometrial cancer cells. FOXC1 was determined as a target gene of miR‐204, and two binding sites in the 3′‐untranslated region were validated by dual luciferase reporter assay. FOXC1 expression was inversely related to miR‐204 expression in EEC. Functional analysis revealed the involvement of FOXC1 in migration and invasion of HEC1A cells. Our results present dysfunctional miRNAs in endometrial cancer and identify a crucial role for miR‐204‐FOXC1 interaction in endometrial cancer progression. This miRNA signature offers a potential biomarker for predicting EEC outcomes, and targeting of these cancer progression‐ and metastasis‐related miRNAs offers a novel potential therapeutic strategy for the disease.

Dysregulated microRNAs and their predicted targets associated with endometrioid endometrial adenocarcinoma in Hong Kong women

The objective of this study, a parallel study to global gene expression profiling, was to identify dysregulated microRNAs (miRNAs) associated with endometrioid endometrial adenocarcinoma (EEC), examine their correlation with clinico‐pathological characteristics and identify predicted target genes of the dysregulated miRNAs. Using real‐time quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR), profiling of miRNA expression was performed in 30 EECs and 22 normal counterparts in which genome‐wide gene expression had been previously profiled and reported. Clustering analysis identified 30 miRNAs which were significantly dysregulated in EEC. The expression of a sub‐group of miRNAs was significantly correlated with clinico‐pathological characteristics including stage, myometrial invasion, recurrence and lymph node involvement. By searching for predicted miRNA targets that were linked to the dysregulated genes previously identified, 68 genes were predicted as candidate targets of these 30 dysregulated miRNAs. miR‐205 was significantly overexpressed in EECs compared with normal controls. After transfection of a miR‐205 inhibitor, the expression of miR‐205 in endometrial cancer cell line RL95‐2 cells decreased whereas its predicted target gene, JPH4, showed increased protein expression. JPH4 seems to be a real miR‐205 target in vitro and in vivo, and a candidate tumor suppressor gene in EEC. Based on this study in EEC, miRNAs predicted to be involved in tumorigenesis and tumor progression have been identified and placed in the context of the transcriptome of EEC. This work provides a framework on which further research into novel diagnosis and treatment of EEC can be focused.

Identification of molecular markers and signaling pathway in endometrial cancer in Hong Kong Chinese women by genome-wide gene expression profiling

Endometrial cancer is the third most common gynecologic malignancy and the ninth most common malignancy for females overall in Hong Kong. Approximately 80% or more of these cancers are endometrioid endometrial adenocarcinomas. The aim of this study was to reveal genes contributing to the development of endometrioid endometrial cancer, which may impact diagnosis, prognosis and treatment of the disease. Whole-genome gene expression analysis was completed for a set of 55 microdissected sporadic endometrioid endometrial adenocarcinomas and 29 microdissected normal endometrium specimens using the Affymetrix Human U133 Plus 2.0 oligonucleotide microarray. Selected genes of interest were validated by quantitative real-time-polymerase chain reaction (qRT-PCR). Pathway analysis was performed to reveal gene interactions involved in endometrial tumorigenesis. Unsupervised hierarchical clustering displayed a distinct separation between the endometrioid adenocarcinomas and normal endometrium samples. Supervised analysis identified 117 highly differentially regulated genes (⩾4.0-fold change), which distinguished the endometrial cancer specimens from normal endometrium. Twelve novel genes including DKK4, ZIC1, KIF1A, SAA2, LOC16378, ALPP2, CCL20, CXCL5, BST2, OLFM1, KLRC1 and MBC45780 were deregulated in the endometrial cancer, and further validated in an independent set of 56 cancer and 29 normal samples using qRT-PCR. In addition, 10 genes were differentially regulated in late-stage cancer, as compared to early-stage disease, and may be involved in tumor progression. Pathway analysis of the expression data from this tumor revealed an interconnected network consisting of 21 aberrantly regulated genes involved in angiogenesis, cell proliferation and chromosomal instability. The results of this study highlight the molecular features of endometrioid endometrial cancer and provide insight into the events underlying the development and progression of endometrioid endometrial cancer.

Dysregulated microRNAs in the pathogenesis and progression of cervical neoplasm.

MicroRNAs (miRNAs) play an important role in a variety of physiological as well as pathophysiological processes, including carcinogenesis. The aim of this study is to identify a distinct miRNA expression signature for cervical intraepithelial neoplasia (CIN) and to unveil individual miRNAs that may be involved in the development of cervical carcinoma. Expression profiling using quantitative real-time RT-PCR of 202 miRNAs was performed on micro-dissected high-grade CIN (CIN 2/3) tissues and compared to normal cervical epithelium. Unsupervised hierarchical clustering of the miRNA expression pattern displayed a distinct separation between the CIN and normal cervical epithelium samples. Supervised analysis identified 12 highly differentially regulated miRNAs, including miR-518a, miR-34b, miR-34c, miR-20b, miR-338, miR-9, miR-512-5p, miR-424, miR-345, miR-10a, miR-193b and miR-203, which distinguished the high-grade CIN specimens from normal cervical epithelium. This miRNA signature was further validated by an independent set of high-grade CIN cases. The same characteristic signature can also be used to distinguish cervical squamous cell carcinoma from normal controls. Target prediction analysis revealed that these dysregulated miRNAs mainly control apoptosis signaling pathways and cell cycle regulation. These findings contribute to understanding the role of microRNAs in the pathogenesis and progression of cervical neoplasm at the molecular level.

Risk Association between Human Leukocyte Antigen–A Allele and High-Risk Human Papillomavirus Infection for Cervical Neoplasia in Chinese Women

To examine the association between human leukocyte antigen-A (HLA-A) allele polymorphism, human papillomavirus (HPV) infection, and risk for cervical neoplasia in Chinese women, 263 patients (155 with cervical intraepithelial neoplasia [CIN] II/III and 108 with invasive cervical cancer [ICC]) were compared with 572 controls. Overall, regardless of HPV status, a decreased risk for ICC was observed for patients with A*0207/0215N or A*2402, and an increased risk was observed for patients with A*1104. The protective association of A*0207/0215N was reproduced in HPV-16-positive patients with ICC, but not in subgroups infected with other HPV types. The risk association between A*1104 and both HPV-16 and HPV-18 was reproduced in the subgroups with CIN III/ICC. The protective association between A*2402 and HPV-16, HPV-18, HPV-52, and HPV-58 was consistently observed in all subgroups with CIN III/ICC, suggesting a linkage with a general antioncogenic genetic factor. The results of the present study indicate that HLA-A polymorphism is one of the host genetic factors that alter the risk for the development of cervical cancer in Chinese women.

CHD5 Downregulation Associated with Poor Prognosis in Epithelial Ovarian Cancer

Background: The CHD5 gene located on 1p36 encodes a protein – chromodomain helicase DNA-binding protein 5. CHD5 has been shown to be a tumor suppressor gene candidate. This study investigated the involvement of CHD5 in ovarian cancer and its clinicopathological significance. Methods: CHD5 expression in ovarian cancer and its counterpart were determined by quantitative RT-PCR. The correlation of CHD5 expression to clinicopathological features of the tumor was analyzed. Results: CHD5 expression was downregulated by at least twofold in 32 of 72 (41%) invasive epithelial ovarian carcinomas when compared to 12 controls in Hong Kong Chinese women. CHD5 downregulation was correlated to clinical status (p < 0.05), but not to patient age, tumor type and grade, recurrence and clinical stage (p > 0.05). Survival analysis showed that patients with CHD5 downregulation in their tumors were associated with shorter disease-free and total survival times compared to those without CHD5 downregulation (p < 0.05). Cox proportional-hazards regression analysis indicated that downregulation of CHD5 is an independent adverse prognostic factor in ovarian cancer. Conclusion: This study shows that CHD5 is downregulated in a certain number of ovarian cancers and appears to be an adverse predictor candidate of ovarian cancer disease-free and total survival.

Cancer cell-derived lymphotoxin mediates reciprocal tumour-stromal interactions in human ovarian cancer by inducing CXCL11 in fibroblasts.

We have investigated the role of cytokine lymphotoxin in tumour–stromal interactions in human ovarian cancer. We found that lymphotoxin overexpression is commonly shared by the cancer cells of various ovarian cancer subtypes, and lymphotoxin‐beta receptor (LTBR) is expressed ubiquitously in both the cancer cells and cancer‐associated fibroblasts (CAFs). In monoculture, we showed that ovarian cancer cells are not the major lymphotoxin‐responsive cells. On the other hand, our co‐culture studies demonstrated that the cancer cell‐derived lymphotoxin induces chemokine expression in stromal fibroblasts through LTBR–NF‐κB signalling. Amongst the chemokines being produced, we found that fibroblast‐secreted CXCL11 promotes proliferation and migration of ovarian cancer cells via the chemokine receptor CXCR3. CXCL11 is highly expressed in CAFs in ovarian cancer biopsies, while CXCR3 is found in malignant cells in primary ovarian tumours. Additionally, the overexpression of CXCR3 is significantly associated with the tumour grade and lymph node metastasis of ovarian cancer, further supporting the role of CXCR3, which interacts with CXCL11, in promoting growth and metastasis of human ovarian cancer. Taken together, these results demonstrated that cancer‐cell‐derived lymphotoxin mediates reciprocal tumour–stromal interactions in human ovarian cancer by inducing CXCL11 in fibroblasts. Our findings suggest that lymphotoxin–LTBR and CXCL11–CXCR3 signalling represent therapeutic targets in ovarian cancer. Copyright

Diclofenac-induced embryotoxicity is associated with increased embryonic 8-isoprostaglandin F2α level in rat whole embryo culture

Diclofenac is embryotoxic in animals but the mechanism of its embryotoxicity is not fully understood. We postulated that diclofenac-induced embryotoxicity might be related to free oxygen radical damage. Rat embryos were explanted at 9.5 days gestation and cultured in medium containing various concentrations of diclofenac. The direct effect of diclofenac on embryonic free oxygen radical content was evaluated by measuring the 8-isoprostaglandin F2alpha level. We found that embryos exposed to high concentrations of diclofenac (7.5 and 15.0 micro g/ml) had significantly higher levels of 8-isoprostaglandin F2alpha (2.85 and 3.50 pg/mm embryonic crown-rump length, respectively) than the control group (1.73 pg/mm, P<0.05), while the 8-isoprostaglandin F2alpha level in embryos exposed to low concentration of diclofenac (1.5 micro g/ml) was 2.54 pg/mm, which was not significantly different from the control group. Median embryo morphologic score was also lower in the high concentration group compared to the control group (39.0 versus 44.0, P<0.05). These results suggest that diclofenac-induced embryopathy may be related to free oxygen radical damage.

Abstract 5047: Dysregulated microRNAs in cervical neoplasm

MicroRNAs (miRNAs) play an important role in a variety of physiological processes as well as pathophysiological processes, including cancinogenesis. This study was to identify a distinct miRNA expression signature for cervical intraepithelial neoplasia (CIN) and to reveal individual miRNAs that may be involved in the development of cervical carcinoma. Expression profiling using quantitative real-time RT-PCR of 202 miRNAs was performed on micro-dissected high-grade CIN (CIN 2/3) tissues as compared to normal cervical epithelium. The expression of 12 miRNAs including miR-518a, miR-34b, miR-34c, miR-20b, miR-338, miR-9, miR-512-5p, miR-424, miR-345, miR-10a, miR-193b and miR-203 were significantly different between high-grade CIN and normal epithelium. This miRNA signature was further validated by an independent set of high-grade CIN tissue. The same characteristic signature can also be used to distinguish cervical squamous cell carcinoma (SCC) from normal controls. Target prediction analysis revealed that these dysregulated miRNAs control the apoptosis signaling pathway and cell cycle regulation. These findings contribute to understanding of the pathogenesis of cervical carcinoma at the molecular level. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5047. doi:1538-7445.AM2012-5047

Clinical use of laparoscopic ultrasonography in detecting nodal metastasis in advanced-stage cervical carcinoma

To assess the clinical use of a laparoscopic ultrasound scan (LUS) to identify pelvic and para‐aortic node metastasis in patients with advanced‐stage cervical cancer.