Rosemarie Schmandt

Dicer, Drosha, and outcomes in patients with ovarian cancer.

BACKGROUND We studied Dicer and Drosha, components of the RNA-interference machinery, in ovarian cancer. METHODS We measured messenger RNA (mRNA) levels of Dicer and Drosha in specimens of invasive epithelial ovarian cancer from 111 patients, using a quantitative reverse-transcriptase-polymerase-chain-reaction assay, and compared the results with clinical outcomes. Validation was performed with the use of published microarray data from cohorts of patients with ovarian, breast, and lung cancer. Mutational analyses of genomic DNA from the Dicer and Drosha genes were performed in a subgroup of ovarian-cancer specimens. Dicer-dependent functional assays were performed by means of in vitro transfection with small interfering RNA (siRNA) and short hairpin RNA (shRNA). RESULTS Levels of Dicer and Drosha mRNA correlated with the levels of expression of the corresponding protein and were decreased in 60% and 51% of ovarian-cancer specimens, respectively. Low Dicer expression was significantly associated with advanced tumor stage (P=0.007), and low Drosha expression with suboptimal surgical cytoreduction (P=0.02). Cancer specimens with both high Dicer expression and high Drosha expression were associated with increased median survival (>11 years, vs. 2.66 years for other subgroups; P<0.001). We found three independent predictors of reduced disease-specific survival in multivariate analyses: low Dicer expression (hazard ratio, 2.10; P=0.02), high-grade histologic features (hazard ratio, 2.46; P=0.03), and poor response to chemotherapy (hazard ratio, 3.95; P<0.001). Poor clinical outcomes among patients with low Dicer expression were validated in additional cohorts of patients. Rare missense mutations were found in the Dicer and Drosha genes, but their presence or absence did not correlate with the level of expression. Functional assays indicated that gene silencing with shRNA, but not siRNA, may be impaired in cells with low Dicer expression. CONCLUSIONS Our findings indicate that levels of Dicer and Drosha mRNA in ovarian-cancer cells have associations with outcomes in patients with ovarian cancer.

Therapeutic EphA2 Gene Targeting In vivo Using Neutral Liposomal Small Interfering RNA Delivery

Inducing destruction of specific mRNA using small interfering RNA (siRNA) is a powerful tool in analysis of protein function, but its use as a therapeutic modality has been limited by inefficient or impractical delivery systems. We have used siRNA incorporated into the neutral liposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) for efficient in vivo siRNA delivery. In nude mice bearing i.p. ovarian tumors, nonsilencing siRNA tagged with the fluorochrome Alexa 555 was encapsulated into DOPC liposomes and shown to be taken up by the tumor as well as many major organs. Furthermore, DOPC-encapsulated siRNA targeting the oncoprotein EphA2 was highly effective in reducing in vivo EphA2 expression 48 hours after a single dose as measured by both Western blot and immunohistochemistry. Therapy experiments in an orthotopic mouse model of ovarian cancer were initiated 1 week after injection of either HeyA8 or SKOV3ip1 cell lines. Three weeks of treatment with EphA2-targeting siRNA-DOPC (150 microg/kg twice weekly) reduced tumor growth when compared with a nonsilencing siRNA (SKOV3ip1: 0.35 versus 0.70 g; P = 0.020; HeyA8: 0.98 versus 1.51 g; P = 0.16). When EphA2-targeting siRNA-DOPC was combined with paclitaxel, tumor growth was dramatically reduced compared with treatment with paclitaxel and a nonsilencing siRNA (SKOV3ip1: 0.04 versus 0.22 g; P < 0.001; HeyA8: 0.21 versus 0.84 g; P = 0.0027). These studies show the feasibility of siRNA as a clinically applicable therapeutic modality.

Selection of Potential Markers for Epithelial Ovarian Cancer with Gene Expression Arrays and Recursive Descent Partition Analysis

Purpose: Advanced-stage epithelial ovarian cancer has a poor prognosis with long-term survival in less than 30% of patients. When the disease is detected in stage I, more than 90% of patients can be cured by conventional therapy. Screening for early-stage disease with individual serum tumor markers, such as CA125, is limited by the fact that no single marker is up-regulated and shed in adequate amounts by all ovarian cancers. Consequently, use of multiple markers in combination might detect a larger fraction of early-stage ovarian cancers. Experimental Design: To identify potential candidates for novel markers, we have used Affymetrix human genome arrays (U95 series) to analyze differences in gene expression of 41,441 known genes and expressed sequence tags between five pools of normal ovarian surface epithelial cells (OSE) and 42 epithelial ovarian cancers of different stages, grades, and histotypes. Recursive descent partition analysis (RDPA) was performed with 102 probe sets representing 86 genes that were up-regulated at least 3-fold in epithelial ovarian cancers when compared with normal OSE. In addition, a panel of 11 genes known to encode potential tumor markers [mucin 1, transmembrane (MUC1), mucin 16 (CA125), mesothelin, WAP four-disulfide core domain 2 (HE4), kallikrein 6, kallikrein 10, matrix metalloproteinase 2, prostasin, osteopontin, tetranectin, and inhibin] were similarly analyzed. Results: The 3-fold up-regulated genes were examined and four genes [Notch homologue 3 (NOTCH3), E2F transcription factor 3 (E2F3), GTPase activating protein (RACGAP1), and hematological and neurological expressed 1 (HN1)] distinguished all tumor samples from normal OSE. The 3-fold up-regulated genes were analyzed using RDPA, and the combination of elevated claudin 3 (CLDN3) and elevated vascular endothelial growth factor (VEGF) distinguished the cancers from normal OSE. The 11 known markers were analyzed using RDPA, and a combination of HE4, CA125, and MUC1 expression could distinguish tumor from normal specimens. Expression at the mRNA level in the candidate markers was examined via semiquantitative reverse transcription-PCR and was found to correlate well with the array data. Immunohistochemistry was performed to identify expression of the genes at the protein level in 158 ovarian cancers of different histotypes. A combination of CLDN3, CA125, and MUC1 stained 157 (99.4%) of 158 cancers, and all of the tumors were detected with a combination of CLDN3, CA125, MUC1, and VEGF. Conclusions: Our data are consistent with the possibility that a limited number of markers in combination might identify >99% of epithelial ovarian cancers despite the heterogeneity of the disease.

Focal Adhesion Kinase Targeting Using In vivo Short Interfering RNA Delivery in Neutral Liposomes for Ovarian Carcinoma Therapy

Purpose: Focal adhesion kinase (FAK) plays a critical role in ovarian cancer cell survival and in various steps in the metastatic cascade. Based on encouraging in vitro results with FAK silencing, we examined the in vivo therapeutic potential of this approach using short interfering RNA (siRNA) in the neutral liposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). Experimental Design: Therapy experiments of FAK siRNA with or without docetaxel were done using human ovarian cancer cell lines SKOV3ip1, HeyA8, and HeyA8MDR in nude mice. Additional experiments with a cisplatin-resistant cell line (A2780-CP20) were also done. Assessments of angiogenesis (CD31), cell proliferation (proliferating cell nuclear antigen), and apoptosis (terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling) were done using immunohistochemical analysis. Results: A single dose of FAK siRNA-DOPC was highly effective in reducing in vivo FAK expression for up to 4 days as assayed by Western blot and immunohistochemical analysis. Therapy experiments were started 1 week after injection of the ovarian cancer cells. Treatment with FAK siRNA-DOPC (150 μg/kg twice weekly) reduced mean tumor weight by 44% to 72% in the three cell lines compared with the control group (Ps < 0.05 for HeyA8, A2780-CP20, and SKOV3ip1). When FAK siRNA-DOPC was combined with docetaxel, there was even greater reduction in mean tumor weight in all models (all Ps < 0.05). Similar results were observed in combination with cisplatin. Treatment with FAK siRNA-DOPC plus docetaxel resulted in decreased microvessel density, decreased expression of vascular endothelial growth factor and matrix metalloproteinase-9, and increased apoptosis of tumor-associated endothelial cells and tumor cells. Conclusions: Taken together, these findings suggest that FAK siRNA-DOPC plus docetaxel or platinum might be a novel therapeutic approach against ovarian cancer.

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.

Gene alterations identified by expression profiling in tumor-associated endothelial cells from invasive ovarian carcinoma.

Therapeutic strategies based on antiangiogenic approaches are beginning to show great promise in clinical studies. However, full realization of these approaches requires identification of key differences in gene expression between endothelial cells from tumors versus their normal counterparts. Here, we examined gene expression differences in purified endothelial cells from 10 invasive epithelial ovarian cancers and 5 normal ovaries using Affymetrix U133 Plus 2.0 microarrays. More than 400 differentially expressed genes were identified in tumor-associated endothelial cells. We selected and validated 23 genes that were overexpressed by 3.6- to 168-fold using real-time reverse transcription-PCR and/or immunohistochemistry. Among these, the polycomb group protein enhancer of Zeste homologue 2 (EZH2), the Notch ligand Jagged1, and PTK2 were elevated 3- to 4.3-fold in tumor-associated endothelial cells. Silencing these genes individually with small interfering RNA blocked endothelial cell migration and tube formation in vitro. The present study shows that tumor and normal endothelium differ at the molecular level, which may have significant implications for the development of antiangiogenic therapies.

Understanding obesity and endometrial cancer risk: opportunities for prevention

Worldwide, obesity has become a major public health crisis. Overweight and obesity not only increase the risk of cardiovascular disease and type-2 diabetes mellitus but also are now known risk factors for a variety of cancer types. Among all cancers, increasing body mass index is associated most strongly with endometrial cancer incidence and death. The molecular mechanisms underlying how adipose tissue and obesity contribute to the pathogenesis of endometrial cancer are becoming better understood and have revealed a number of rational strategies, both behavioral and pharmaceutical, for the prevention of both primary and recurrent disease.

Omental adipose tissue-derived stromal cells promote vascularization and growth of endometrial tumors.

Purpose: Adipose tissue contains a population of tumor-tropic mesenchymal progenitors, termed adipose stromal cells (ASC), which engraft in neighboring tumors to form supportive tumor stroma. We hypothesized that intra-abdominal visceral adipose tissue may contain a uniquely tumor-promoting population of ASC to account for the relationship between excess visceral adipose tissue and mortality of intra-abdominal cancers. Experimental Design: To investigate this, we isolated and characterized ASC from intra-abdominal omental adipose tissue (O-ASC) and characterized their effects on endometrial cancer progression as compared with subcutaneous adipose-derived mesenchymal stromal cells (SC-ASC), bone marrow–derived mesenchymal stromal cells (BM-MSC), and lung fibroblasts. To model chronic recruitment of ASC by tumors, cells were injected metronomically into mice bearing Hec1a xenografts. Results: O-ASC expressed cell surface markers characteristic of BM-MSC and differentiated into mesenchymal lineages. Coculture with O-ASC increased endometrial cancer cell proliferation in vitro. Tumor tropism of O-ASC and SC-ASC for human Hec1a endometrial tumor xenografts was comparable, but O-ASC more potently promoted tumor growth. Compared with tumors in SC-ASC–injected mice, tumors in O-ASC–injected mice contained higher numbers of large tortuous desmin-positive blood vessels, which correlated with decreased central tumor necrosis and increased tumor cell proliferation. O-ASC exhibited enhanced motility as compared with SC-ASC in response to Hec1a-secreted factors. Conclusions: Visceral adipose tissue contains a population of multipotent MSCs that promote endometrial tumor growth more potently than MSCs from subcutaneous adipose tissue. We propose that O-ASCs recruited to tumors express specific factors that enhance tumor vascularization, promoting survival and proliferation of tumor cells. Clin Cancer Res; 18(3); 771–82. ©2011 AACR.

Expression of c-ABL, c-KIT, and platelet-derived growth factor receptor-β in ovarian serous carcinoma and normal ovarian surface epithelium

Tyrosine kinases, such as c‐KIT, c‐ABL, and platelet‐derived growth factor‐beta (PDGFR‐β), are important regulators of cell growth. Highly potent and selective inhibitors of tyrosine kinases are being investigated as alternatives to standard chemotherapy. One such inhibitor, imatinib mesylate, is being used to treat gastrointestinal stromal tumors and chronic myelogenous leukemia. Ovarian carcinomas frequently develop resistance to conventional chemotherapeutic agents. Immunohistochemical expression of c‐ABL, PDGFR‐β, and c‐KIT was evaluated in ovarian carcinomas to determine whether treatment with imatinib mesylate might be feasible.

Exosomal transfer of stroma-derived miR21 confers paclitaxel resistance in ovarian cancer cells through targeting APAF1

Advanced ovarian cancer usually spreads to the visceral adipose tissue of the omentum. However, the omental stromal cell-derived molecular determinants that modulate ovarian cancer growth have not been characterized. Here, using next-generation sequencing technology, we identify significantly higher levels of microRNA-21 (miR21) isomiRNAs in exosomes and tissue lysates isolated from cancer-associated adipocytes (CAAs) and fibroblasts (CAFs) than in those from ovarian cancer cells. Functional studies reveal that miR21 is transferred from CAAs or CAFs to the cancer cells, where it suppresses ovarian cancer apoptosis and confers chemoresistance by binding to its direct novel target, APAF1. These data suggest that the malignant phenotype of metastatic ovarian cancer cells can be altered by miR21 delivered by exosomes derived from neighbouring stromal cells in the omental tumour microenvironment, and that inhibiting the transfer of stromal-derived miR21 is an alternative modality in the treatment of metastatic and recurrent ovarian cancer.