Adnan R. Munkarah

Regulation of Tumor Angiogenesis by EZH2

Although VEGF-targeted therapies are showing promise, new angiogenesis targets are needed to make additional gains. Here, we show that increased Zeste homolog 2 (EZH2) expression in either tumor cells or in tumor vasculature is predictive of poor clinical outcome. The increase in endothelial EZH2 is a direct result of VEGF stimulation by a paracrine circuit that promotes angiogenesis by methylating and silencing vasohibin1 (vash1). Ezh2 silencing in the tumor-associated endothelial cells inhibited angiogenesis mediated by reactivation of VASH1, and reduced ovarian cancer growth, which is further enhanced in combination with ezh2 silencing in tumor cells. Collectively, these data support the potential for targeting ezh2 as an important therapeutic approach.

Epigenetic silencing of TH1-type chemokines shapes tumour immunity and immunotherapy

Epigenetic silencing including histone modifications and DNA methylation is an important tumorigenic mechanism. However, its role in cancer immunopathology and immunotherapy is poorly understood. Using human ovarian cancers as our model, here we show that enhancer of zeste homologue 2 (EZH2)-mediated histone H3 lysine 27 trimethylation (H3K27me3) and DNA methyltransferase 1 (DNMT1)-mediated DNA methylation repress the tumour production of T helper 1 (TH1)-type chemokines CXCL9 and CXCL10, and subsequently determine effector T-cell trafficking to the tumour microenvironment. Treatment with epigenetic modulators removes the repression and increases effector T-cell tumour infiltration, slows down tumour progression, and improves the therapeutic efficacy of programmed death-ligand 1 (PD-L1; also known as B7-H1) checkpoint blockade and adoptive T-cell transfusion in tumour-bearing mice. Moreover, tumour EZH2 and DNMT1 are negatively associated with tumour-infiltrating CD8+ T cells and patient outcome. Thus, epigenetic silencing of TH1-type chemokines is a novel immune-evasion mechanism of tumours. Selective epigenetic reprogramming alters the T-cell landscape in cancer and may enhance the clinical efficacy of cancer therapy.

Metformin: An Emerging New Therapeutic Option for Targeting Cancer Stem Cells and Metastasis

Metastasis is an intricate process by which a small number of cancer cells from the primary tumor site undergo numerous alterations, which enables them to form secondary tumors at another and often multiple sites in the host. Transition of a cancer cell from epithelial to mesenchymal phenotype is thought to be the first step in the progression of metastasis. Recently, the recognition of cancer stem cells has added to the perplexity in understanding metastasis, as studies suggest cancer stem cells to be the originators of metastasis. All current and investigative drugs have been unable to prevent or reverse metastasis, as a result of which most metastatic cancers are incurable. A potential drug that can be considered is metformin, an oral hypoglycemic drug. In this review we discuss the potential of metformin in targeting both epithelial to mesenchymal transition and cancer stem cells in combating cancer metastases.

Clinical trials and progress with paclitaxel in ovarian cancer.

Paclitaxel is a front-line agent for ovarian cancer chemotherapy, along with the platinum agents. Derived from the Pacific yew tree, Taxus brevifolia, paclitaxel has covered significant ground from the initial discovery of its antineoplastic properties to clinical applications in many forms of human cancers, including ovarian cancer. Although much has been published about the unique mechanism of action of this agent, several issues remain to be resolved. Finding the appropriate dosage schedule for paclitaxel in chemo-naïve and recurrent ovarian cancer, defining the role of paclitaxel in maintenance chemotherapy, and elucidating the mechanisms of taxane resistance are areas of intense research. Newer forms of taxanes are being manufactured to avoid troublesome adverse effects and to improve clinical efficacy. These issues are reviewed in detail in this paper with an emphasis on clinically relevant evidence-based information.

The Role of Frozen Section in Surgical Staging of Low Risk Endometrial Cancer

Background The role of frozen section (FS) in intraoperative decision making for surgical staging of endometrial cancer is controversial. Objective of this study is to assess the agreement rate between the FS and paraffin section (PS); and the potential impact of the role of FS in the intra-operative decision making for the complete surgical staging in low risk endometrial cancer. Methods This is a retrospective analysis of patients diagnosed with intra-operative FS stage I, grade I or II endometrial cancer from 1995–2004. FS results were compared with final pathology results with regard to tumor grade, depth of myometrial invasion, cervical involvement, lymphovascular invasion, and lymph node involvement. Agreement statistic with kappa was calculated using SPSS statistical software. Categorical variables were tested using chi-square test with p value of ≤0.05 being statistically significant. Results Of the 457 patients with endometrial cancer, 146 were evaluated by intra-operative FS and met inclusion criteria. FS results were in disagreement with permanent section in 35% for the grade (kappa 0.58, p = 0.003), 28% for depth of myometrial invasion (kappa 0.61, p<0.0001), 13% for cervical involvement (kappa 0.78, p = 0.002), and 32% for lymphovascular invasion (kappa 0.6, p = 0.01). Permanent pathology upstaged 31.9% & 23.2% of FS stage IA, & IB specimen respectively. Lymph node dissection was done in 56.8%. Lymph node metastasis was identified in 8.4%. Use of intraoperative FS would have resulted in suboptimal surgical treatment in 13% stage IA and 6.6% of stage IB patients respectively by foregoing lymphadenectomy. Conclusion A significant number of patients with low risk endometrial cancer by FS were upstaged and upgraded on final pathology. Before placing absolute reliance on intraoperative FS to undertake complete surgical staging, the inherent limitation of the same in predicting final stage and grade highlighted by our data need to be carefully considered.

Sensitization of ovarian cancer cells to cisplatin by genistein: the role of NF-kappaB

BackgroundPlatinum-resistance (PR) continues to be a major problem in the management of epithelial ovarian cancer (EOC). Response to various chemotherapeutic agents is poor in patients deemed PR. Genistein, a soy isoflavone has been shown to enhance the effect of chemotherapy in prostate and pancreatic cancer cells in vitro and in vivo by reversing chemo-resistance phenotype. The goal of this study was to investigate the effects of combination therapy with genistein and cisplatin as well as other cytotoxic conventional chemotherapeutic agents in platinum-sensitive (PS) and resistant EOC cells.MethodsThe PS human ovarian cancer cell line A2780 and its PR clone C200 cells were pretreated with genistein, followed by the combination of genistein and either cisplatin, taxotere or gemcitabine. Cell survival and apoptosis was assessed by MTT and histone-DNA ELISA. Electrophoretic mobility shift assay (EMSA) was used to evaluate NF-κB DNA binding activity. Western blot analysis was performed with antibodies to Bcl-2, Bcl-xL, survivin, c-IAP and PARP.ResultsReduction in cell viability, and corresponding induction of apoptosis was observed with genistein pretreatment followed by combination treatment with each of the drugs in both cell lines. The PS cell line was pretreated for 24 hours; in contrast, the PR cell line required 48 hours pretreatment to achieve a response. The anti-apoptotic genes c-IAP1, Bcl-2, Bcl-xL, survivin and NF-κB DNA binding activity were all found to be down-regulated in the combination groups.ConclusionThis study convincingly demonstrated that the current strategy can be translated in a pre-clinical animal model, and thus it should stimulate future clinical trial for the treatment of drug-resistant ovarian cancer.

Patterns of loss of heterozygosity at 10q23.3 and microsatellite instability in endometriosis, atypical endometriosis, and ovarian carcinoma arising in association with endometriosis.

Summary: Genetic aberrations, such as loss of heterozygosity (LOH) and mutations leading to functional inactivation of the PTEN tumor suppressor gene, located on chromosome 10q23.3, have been shown to be associated with approximately one third of ovarian adenocarcinomas. In addition, microsatellite instability (MSI) leading to the functional inactivation of the PTEN gene has also been reported for ovarian adenocarcinomas with frequencies varying from 6 to 37%. However, the frequency of PTEN gene abnormalities has not been well studied or evaluated in lesions such as typical and atypical endometriosis. The aim of this study was to investigate a possible sequential progression from endometriosis through atypical endometriosis to ovarian carcinoma by assessing LOH at 10q23.3 and MSI in those entities. Genomic DNA was analyzed for LOH and MSI at 3 loci on chromosome 10, using polymerase chain reaction amplification. Significant differences in LOH were seen between endometriosis (4.3%) and ovarian carcinoma (23.5%) at D10S608. The differences at the other 2 loci were not significant. A high frequency of MSI was found in endometriosis (82.6%) and atypical endometriosis (75%); however, the differences were not significant. These results suggest that LOH at D105608 may possibly be an important molecular event in the progression of endometriosis to carcinoma. This study highlights that endometriosis and atypical endometriosis might act as precursor lesions that have the potential to progress into ovarian adenocarcinoma.

Cyclooxygenase-2 is expressed in human fibroblasts isolated from intraperitoneal adhesions but not from normal peritoneal tissues

OBJECTIVE To determine whether the COX-2 gene is expressed in human fibroblasts isolated from normal peritoneal and adhesion tissues. DESIGN Prospective experimental study. SETTING University medical center. PATIENT(S) Five patients undergoing laparotomy for pelvic pain. Primary cultures of fibroblasts were taken from both peritoneum and adhesion tissues. INTERVENTION(S) Hypoxia treatment of the primary cultured fibroblasts. MAIN OUTCOME MEASURES We used the multiplex reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry techniques to determine whether COX-2 mRNA and its protein were present in normal peritoneal and adhesion fibroblasts from the same patients. Total RNA was extracted from cultured fibroblasts and subjected to multiplex RT-PCR to detect the presence of COX-2 mRNA in these cells. Cultured fibroblasts from all tissues were also fixed on slides and stained with COX-2 monoclonal antibody labeled with immunofluorescence. RESULT(S) COX-2 mRNA and its protein were absent in normal peritoneal fibroblasts from all five subjects but were present in adhesion fibroblasts from the same patients, as indicated by the multiplex RT-PCR and immunohistochemistry techniques. Hypoxia treatment significantly induced the mRNA and COX-2 protein levels in normal peritoneal fibroblasts to levels seen in adhesion fibroblasts under normoxic conditions. However, hypoxia had no effects on COX-2 expression by adhesion fibroblasts. CONCLUSION(S) Adhesion fibroblasts develop a specific phenotype, an adhesion phenotype, which is in part characterized by the expression of COX-2. The expression of COX-2 mRNA in adhesion fibroblasts and the induction of COX-2 in peritoneal fibroblasts in response to hypoxia indicate a possible inflammatory response. Regulation of COX-2 may alter peritoneal healing and may provide the opportunity to reduce postoperative adhesion development.

MicroRNA-101 Inhibits Growth of Epithelial Ovarian Cancer by Relieving Chromatin-Mediated Transcriptional Repression of p21 waf1/cip1

ABSTRACTPurposeMicroRNA-101 (miR-101) expression is negatively associated with tumor growth and proliferation in several solid epithelial cancers. Enhancer of zeste homolog 2 (EzH2) appears to be a functional target of miR-101. We explore the role of miR-101 and its interaction with EzH2 in epithelial ovarian carcinoma (EOC).MethodsIn situ hybridization (ISH) for miR-101 was performed on EOC patient tissues and normal controls. EOC cell lines were transfected with miR-101 and subjected to growth analysis and clonogenic assays. Cell motility was assessed by Boyden chamber and wound-healing assays. P21waf1/cip1 and EzH2 interaction was assessed by Chromatin Immunoprecipitation (ChIP) assay in MDAH-2774 cells. SCID mice were assessed for tumor burden after injection with miR-101 or control vector-treated MDAH-2774 cells.ResultsISH analysis revealed a decrease in miR-101 expression in EOC compared with normal tissue. MiR-101 re-expression in EOC cell lines resulted in increased apoptosis, decreased cellular proliferation, invasiveness, and reduced growth of tumor xenografts. CHIP assays revealed that re-expression of miR-101 inhibited the interaction of EzH2 with p21waf1/cip1 promoter.ConclusionsMiR-101 re-expression appears to have antitumor effects, providing a better understanding of the role of miR-101 in EOC.

Prognostic analysis of ovarian cancer associated with endometriosis

OBJECTIVE The objective of the study was to evaluate the prognosis of ovarian cancer arising in endometriosis. STUDY DESIGN We retrospectively compared 42 cases of endometriosis-associated ovarian cancer (EAOC) with 184 cases of ovarian carcinoma without endometriosis (OC). RESULTS The median age in the EAOC group was 52 vs 59 years in OC (P < .05). In comparison with OC, the EAOC patients were more likely to have low-grade (21% vs 8%; P = .04) and early-stage tumors (International Federation of Gynecology and Obstetrics I and II combined) (49% vs 24%; P = .002). Clear cell (21% vs 2%) and endometrioid (14% vs 3%) tumors were more frequent in EAOC, whereas mucinous tumors were more prevalent in OC (P = .001). The median survival (199 vs 62 months) and the 5 year survival (62% vs 51%) were better for EAOC when compared with OC (P = .038). After controlling for age, stage, grade, and treatment, association with endometriosis was not an independent predictor of better survival in ovarian cancer. CONCLUSION As such, EAOC has a much better survival rate than OC. This could be explained by the higher prevalence of early-stage and low-grade tumors in EAOC when compared with OC.