We studied Dicer and Drosha, components of the RNA-interference machinery, in ovarian cancer.
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).
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.
Our findings indicate that levels of Dicer and Drosha mRNA in ovarian-cancer cells have associations with outcomes in patients with ovarian cancer.
Focal adhesion kinase (FAK) overexpression is frequently found in ovarian and other cancers and is predictive of poor clinical outcome. In the current study, we characterized the biological and ...therapeutic effects of a novel FAK inhibitor, TAE226. Taxane-sensitive (SKOV3ip1 and HeyA8) and taxane-resistant (HeyA8-MDR) cell lines were used for in vitro and in vivo therapy experiments using TAE226 alone and in combination with docetaxel. Assessment of cytotoxicity, cell proliferation proliferating cell nuclear antigen (PCNA), angiogenesis (CD31), and apoptosis (terminal nucleotidyl transferase-mediated nick end labeling) were done by immunohistochemistry and immunofluorescence. In vitro, TAE226 inhibited the phosphorylation of FAK at both Y397 and Y861 sites, inhibited cell growth in a time- and dose-dependent manner, and enhanced docetaxel-mediated growth inhibition by 10- and 20-fold in the taxane-sensitive and taxane-resistant cell lines, respectively. In vivo, FAK inhibition by TAE226 significantly reduced tumor burden in the HeyA8, SKOV3ip1, and HeyA8-MDR models (46-64%) compared with vehicle-treated controls. However, the greatest efficacy was observed with concomitant administration of TAE226 and docetaxel in all three models (85-97% reduction, all P values <0.01). In addition, TAE226 alone and in combination with chemotherapy significantly prolonged survival in tumor-bearing mice. Even in larger tumors, combination therapy with TAE226 and docetaxel resulted in tumor regression. The therapeutic efficacy was related to reduced pericyte coverage, induction of apoptosis of tumor-associated endothelial cells, and reduced microvessel density and tumor cell proliferation. The novel FAK inhibitor, TAE226, offers an attractive therapeutic approach in ovarian carcinoma.
Metastasis is a complex, multistep process that begins with the epithelial-mesenchymal transition (EMT). Circulating tumor cells (CTC) are believed to have undergone EMT and thus lack or express low ...levels of epithelial markers commonly used for enrichment and/or detection of such cells. However, most current CTC detection methods target only EpCAM and/or cytokeratin (CK) to enrich epithelial CTCs, resulting in failure to recognize other, perhaps more important, CTC phenotypes that lack expression of these markers. Here, we describe a population of complex aneuploid CTCs that do not express CK or CD45 antigen in patients with breast, ovarian, or colorectal cancer. These cells were not observed in healthy subjects. We show that the primary epithelial tumors were characterized by similar complex aneuploidy, indicating conversion to an EMT phenotype in the captured cells. Collectively, our study provides a new method for highly efficient capture of previously unrecognized populations of CTCs.
Current assays for CTC capture likely miss populations of cells that have undergone EMT. Capture and study of CTCs that have undergone EMT would allow a better understanding of the mechanisms driving metastasis.
The dynamic properties of RNA interference (RNAi) in cancer biology have led investigators to pursue with significant interest its role in tumorigenesis and cancer therapy. We recently reported that ...decreased expression of key RNAi enzymes, Dicer and Drosha, in epithelial ovarian cancers was associated with poor clinical outcome in patients. Dicer expression was also functionally relevant in that targeted silencing was limited with RNAi fragments that require Dicer function compared with those that do not. Together, this and other studies suggest that RNAi machinery expression may affect key pathways in tumorigenesis and cancer biology. Understanding alterations in the functional RNAi machinery is of fundamental importance as we strive to develop novel therapies using RNAi strategies.
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).
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.
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 mug/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.
Taken together, these findings suggest that FAK siRNA-DOPC plus docetaxel or platinum might be a novel therapeutic approach against ovarian cancer.
Vascular endothelial growth factor (VEGF) is critical for angiogenesis and tumor progression; however, its role in endometrial cancer is not fully known. Therefore, we examined the clinical and ...therapeutic significance of VEGF in endometrial carcinoma using patient samples and an endometrioid orthotopic mouse model.
Following Institutional Review Board approval, VEGF expression and microvessel density (MVD) counts were evaluated using immunohistochemistry in 111 invasive endometrioid endometrial cancers by two independent investigators. Results were correlated with clinicopathologic characteristics. For the animal model, Ishikawa or Hec-1A cancer cell lines were injected directly into the uterine horn. Therapy experiments with bevacizumab alone or in combination with docetaxel were done and samples were analyzed for markers of angiogenesis and proliferation.
Of 111 endometrial cancers, high expression of VEGF was seen in 56% of tumors. There was a strong correlation between VEGF expression and MVD (P < 0.001). On multivariate analysis, stage (P = 0.04), grade (P = 0.003), VEGF levels (P = 0.03), and MVD (P = 0.037) were independent predictors of shorter disease-specific survival. In the murine model, whereas docetaxel and bevacizumab alone resulted in 61% to 77% tumor growth inhibition over controls, combination therapy had the greatest efficacy (85-97% inhibition over controls; P < 0.01) in both models. In treated tumors, combination therapy significantly reduced MVD counts (50-70% reduction over controls; P < 0.01) and percent proliferation (39% reduction over controls; P < 0.001).
Increased levels of VEGF and angiogenic markers are associated with poor outcome in endometrioid endometrial cancer patients. Using a novel orthotopic model of endometrioid endometrial cancer, we showed that combination of antivascular therapy with docetaxel is highly efficacious and should be considered for future clinical trials.
Stress can alter immunological, neurochemical and endocrinological functions, but its role in cancer progression is not well understood. Here, we show that chronic behavioral stress results in higher ...levels of tissue catecholamines, greater tumor burden and more invasive growth of ovarian carcinoma cells in an orthotopic mouse model. These effects are mediated primarily through activation of the tumor cell cyclic AMP (cAMP)-protein kinase A (PKA) signaling pathway by the β2 adrenergic receptor (encoded by ADRB2). Tumors in stressed animals showed markedly increased vascularization and enhanced expression of VEGF, MMP2 and MMP9, and we found that angiogenic processes mediated the effects of stress on tumor growth in vivo. These data identify β-adrenergic activation of the cAMP-PKA signaling pathway as a major mechanism by which behavioral stress can enhance tumor angiogenesis in vivo and thereby promote malignant cell growth. These data also suggest that blocking ADRB-mediated angiogenesis could have therapeutic implications for the management of ovarian cancer.
To assess the clinical significance of Aurora-A kinase, a centrosome-regulating serine-threonine kinase, in ovarian carcinoma.
Aurora-A kinase expression was assessed by Western blot (cell lines) or ...immunohistochemistry (high-grade epithelial ovarian cancers), and clinical variables were collected by retrospective chart review. Centrosome amplification was assessed by immunofluorescence in cell lines, and by immunohistochemistry in patient samples.
All ovarian cancer cell lines exhibited significant Aurora-A kinase protein overexpression, and all except A2780-par had centrosome amplification, a characteristic of mitotic dysregulation leading to genomic instability. Fifty-eight of 70 patient samples (82.8%) exhibited Aurora-A kinase overexpression compared with normal ovarian surface epithelium. High Aurora-A kinase expression was strongly associated with supernumerary centrosome count in tumor cells (P<0.001). Tumors with the greatest Aurora-A overexpression (n=24) had decreased patient survival (median survival, 1.44 versus 2.81 years; P=0.01). High Aurora-A expression and suboptimal surgical cytoreduction remained predictors of poor survival (P<0.05) by multivariate analysis.
Aurora-A kinase is overexpressed by a substantial proportion of ovarian cancers and is associated with centrosome amplification and poor survival. It may be a useful prognostic marker and target in ovarian cancer.
Chronic stress is associated with hormonal changes that are known to affect multiple systems, including the immune and endocrine systems, but the effects of stress on cancer growth and progression ...are not fully understood. Here, we demonstrate that human ovarian cancer cells exposed to either norepinephrine or epinephrine exhibit lower levels of anoikis, the process by which cells enter apoptosis when separated from ECM and neighboring cells. In an orthotopic mouse model of human ovarian cancer, restraint stress and the associated increases in norepinephrine and epinephrine protected the tumor cells from anoikis and promoted their growth by activating focal adhesion kinase (FAK). These effects involved phosphorylation of FAKY397, which was itself associated with actin-dependent Src interaction with membrane-associated FAK. Importantly, in human ovarian cancer patients, behavioral states related to greater adrenergic activity were associated with higher levels of pFAKY397, which was in turn linked to substantially accelerated mortality. These data suggest that FAK modulation by stress hormones, especially norepinephrine and epinephrine, can contribute to tumor progression in patients with ovarian cancer and may point to potential new therapeutic targets for cancer management.
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