Mavis S. Fletcher

Effect of Interleukin-8 Gene Silencing With Liposome-Encapsulated Small Interfering RNA on Ovarian Cancer Cell Growth

BACKGROUND Interleukin-8 (IL-8) is a proangiogenic cytokine that is overexpressed in many human cancers. We investigated the clinical and biologic significance of IL-8 in ovarian carcinoma using human samples and orthotopic mouse models. METHODS Tumor expression of IL-8 was assessed by immunohistochemistry among ovarian cancer patients (n = 102) with available clinical and survival data. We examined the effect of IL-8 gene silencing with small interfering RNAs incorporated into neutral liposomes (siRNA-DOPCs), alone and in combination with docetaxel, on in vivo tumor growth, angiogenesis (microvessel density), and tumor cell proliferation in mice (n = 10 per treatment group) bearing orthotopic taxane-sensitive (HeyA8 and SKOV3ip1) and taxane-resistant (SKOV3ip2.TR) ovarian tumors. All statistical tests were two-sided. RESULTS Of the 102 cancer specimens, 43 (42%) had high IL-8 expression and 59 (58%) had low or no IL-8 expression; high IL-8 expression was associated with advanced tumor stage (P = .019), high tumor grade (P = .031), and worse survival (median survival for patients with high vs low IL-8 expression: 1.62 vs 3.79 years; P < .001). Compared with empty liposomes, IL-8 siRNA-DOPC reduced the mean tumor weight by 32% (95% confidence interval [CI] = 14% to 50%; P = .03) and 52% (95% CI = 27% to 78%; P = .03) in the HeyA8 and SKOV3ip1 mouse models, respectively. In all three mouse models, treatment with IL-8 siRNA-DOPC plus the taxane docetaxel reduced tumor growth the most compared with empty liposomes (77% to 98% reduction in tumor growth; P < .01 for all). In the HeyA8 and SKOV3ip1 models, tumors from mice treated with IL-8 siRNA-DOPC alone had lower microvessel density than tumors from mice treated with empty liposomes (HeyA8: 34% lower, 95% CI = 32% to 36% lower [P = .002]; SKOV3ip1: 39% lower, 95% CI = 34% to 44% lower [P = .007]). Compared with empty liposomes, IL-8 siRNA-DOPC plus docetaxel reduced tumor cell proliferation by 35% (95% CI = 25% to 44%; P < .001) and 38% (95% CI = 28% to 48%; P < .001) in the HeyA8 and SKOV3ip1 models, respectively. CONCLUSIONS Increased IL-8 expression is associated with poor clinical outcome in human ovarian carcinoma, and IL-8 gene silencing decreases tumor growth through antiangiogenic mechanisms.

Biological significance of focal adhesion kinase in ovarian cancer: Role in migration and invasion

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that is activated by integrin clustering. There are limited data regarding the functional role of FAK in ovarian cancer migration and invasion. In the current study, FAK expression was evaluated in ovarian cell lines (nontransformed and cancer), 12 benign ovarian samples, and in 79 invasive epithelial ovarian cancers. All three ovarian cancer cell lines overexpressed FAK compared to the nontransformed cells. The dominant-negative construct called FAK-related nonkinase (FRNK) was introduced into two ovarian cancer cell lines (SKOV3 and 222). FRNK promoted FAK dephosphorylation without changing total FAK levels in these cell lines. Furthermore, FRNK decreased the in vitro invasive ability of ovarian cancer cells by 56 to 85% and decreased migration by 52 to 68%. FRNK-transfected cells also displayed poor cell spreading. Immunohistochemical analysis revealed that the surface epithelium from all benign ovarian samples had weak FAK expression. In contrast, 68% of invasive ovarian cancers overexpressed FAK. FAK overexpression was significantly associated with high tumor stage, high tumor grade, positive lymph nodes and presence of distant metastasis (all P values <0.05). FAK overexpression was also associated with shorter overall survival (P = 0.008). Multivariate analysis revealed that FAK overexpression and residual disease >1 cm were independent predictors of poor survival. These data indicate that FAK is overexpressed in most invasive ovarian cancers and plays a functionally significant role in ovarian cancer migration and invasion. Thus, FAK may be an important therapeutic target in ovarian carcinoma.

EphA2 expression is associated with aggressive features in ovarian carcinoma.

Purpose: EphA2 (epithelial cell kinase) is a transmembrane receptor tyrosine kinase that has been implicated in oncogenesis. There are no published data regarding the role of EphA2 in ovarian carcinoma, which is the focus of the present study. Experimental Design: Nontransformed (HIO-180) and ovarian cancer (EG, 222, SKOV3, and A2780-PAR) cell lines were evaluated for EphA2 by Western blot analysis. Five benign ovarian masses, 10 ovarian tumors of low malignant potential, and 79 invasive ovarian carcinomas were also evaluated for EphA2 expression by immunohistochemistry. All samples were scored in a blinded fashion. Univariate and multivariate analyses were used to determine significant associations between EphA2 expression and clinicopathological variables. Results: By Western blot analysis, EG, 222, and SKOV3 cell lines overexpressed EphA2, whereas A2780-PAR and HIO-180 had low to absent EphA2 expression. All of the benign tumors had low or absent EphA2 expression. Among the invasive ovarian carcinomas examined (mean age of patients was 59.2 years), 60 (75.9%) tumors overexpressed EphA2 and the other 19 tumors had negative or minimal EphA2 expression. There was no association of EphA2 overexpression with ascites, likelihood of nodal positivity, pathological subtype, and optimum surgical cytoreduction (residual tumor <1 cm). However, EphA2 overexpression was significantly associated with higher tumor grade (P = 0.02) and advanced stage of disease (P = 0.001). The median survival for patients with tumor EphA2 overexpression was significantly shorter (median, 3.1 years; P = 0.004); the median survival for patients with low or absent EphA2 tumor expression was at least 12 years and has not yet been reached. In multivariate analysis using the Cox proportional hazards model, only volume of residual disease (P < 0.04) and EphA2 overexpression (P < 0.01) were significant and independent predictors of survival. Conclusions: EphA2 overexpression is predictive of aggressive ovarian cancer behavior and may be an important therapeutic target.

The Clinical Relevance of Stromal Matrix Metalloproteinase Expression in Ovarian Cancer

Purpose: Matrix metalloproteinases (MMP) are proteolytic enzymes implicated in cancer progression and metastasis. We sought to determine the role of epithelial (tumor cell–derived) and stromal (host-derived) expression of MMPs in predicting the clinical outcome of patients with epithelial ovarian cancer (EOC). Experimental Design: MMP-2, MMP-9, and membrane type 1 (MT1)-MMP expression was evaluated using immunohistochemistry in 90 invasive EOCs, and samples were scored for epithelial and stromal staining. Results were correlated with clinicopathologic characteristics using univariate and multivariate analyses. Results: High expression of MMP-2, MMP-9, and MT1-MMP in tumor epithelium was detected in 54%, 97%, and 100% of cases, and in stromal compartments, in 38%, 70%, and 38% of cases, respectively. High stromal expression of MMP-2, MMP-9, and MT1-MMP was significantly associated with aggressive features such as high stage, high grade ascites, and positive lymph node status. Kaplan-Meier analysis showed that high epithelial and stromal expression of MMP-2, MMP-9, and MT1-MMP were each significantly associated with shorter disease-specific survival (DSS; P < 0.01). On tree-structured survival analysis, patients with strong epithelial MT1-MMP expression had the shortest DSS, whereas patients with moderate epithelial MT1-MMP and low stromal MMP-9 expression had the longest DSS (P < 0.01). On multivariate analysis, high stromal expression of MMP-9 (P = 0.01) and MT1-MMP (P = 0.04), strong epithelial MT1-MMP (P = 0.01) and high stage (P = 0.04) were independent predictors of poor DSS. Conclusions: Overexpression of stromal MMP-9 and MT1-MMP is independently associated with shorter DSS in EOC. Thus, host-derived MMPs are valuable predictors of clinical outcome in EOC.

HLA class I antigen processing machinery component expression and intratumoral T-cell infiltrate as independent prognostic markers in ovarian carcinoma

Purpose: Defects in the antigen processing machinery (APM) may provide tumor cells with a mechanism to escape immune recognition. The purpose of this study is to determine the clinical significance of APM component down-regulation and tumor-infiltrating T cells in ovarian carcinoma. Experimental Design: After institutional review board approval, tumor samples from 150 patients with invasive epithelial ovarian cancers were examined for TAP1, TAP2, tapasin, HLA class I heavy chain (HLA-HC), β2 microglobulin, and T-cell (CD3+ and CD8+) tumor infiltration using immunohistochemistry. Results: The majority of tumors had either heterogeneous or positive expression of TAP1, TAP2, HLA-HC, and β2 microglobulin (66.7%, 73.3%, 70.7%, and 63.3%, respectively), except tapasin for which 58% of the tumors lacked expression. Furthermore, 67% and 88% of the lesions possessed intratumoral and peritumoral CD3+ or CD8+ cells, respectively. The majority of APM component expression examined was significantly associated with both intratumoral and peritumoral T-cell infiltration (P < 0.05). The expression of APM components and the presence of intratumoral T-cell infiltrates were significantly associated with improved survival (all P ≤ 0.01); however, peritumoral T-cell infiltrates did not significantly affect survival (P = 0.33). APM component down-regulation (P < 0.001), lack of intratumoral T-cell infiltrates (P = 0.03), and suboptimal cytoreduction (P < 0.001) were independent prognostic markers for death from ovarian carcinoma. Conclusion: The negative effect of APM component down-regulation by itself and in combination with absent intratumoral T-cell infiltration on the survival of patients with ovarian carcinoma implies a role for immune escape in addition to immunosurveillance in the clinical course of disease.

EphA2 overexpression is associated with angiogenesis in ovarian cancer

EphA2 is overexpressed in the majority of ovarian cancers and predicts poor clinical outcome. Based on EphA2s emerging role in angiogenesis, we hypothesized that tumors overexpressing EphA2 demonstrate greater microvessel density (MVD) and matrix metalloproteinase (MMP) expression.

The clinical significance of tumor cell-lined vasculature in ovarian carcinoma: implications for anti-vasculogenic therapy.

Vasculogenic mimicry reflects the plasticity of aggressive tumor cells that express vascular cell markers and line tumor vasculature; such has been demonstrated in aggressive ovarian carcinoma. This study measured the clinical significance of tumor cell-lined vasculature in ovarian carcinomas (n = 77), which was detected in 23 (29.8%) tumors. The data show that tumor cell-lined vasculature was associated with aggressive tumor features and with shorter overall survival (p < 0.001). Cox proportional hazards model revealed that tumor cell-lined vasculature (p = 0.002) was independently associated with poor survival. This is the first study demonstrating the clinical implications of tumor cell-lined vasculature in ovarian carcinoma.

Analysis of EphA2 Expression and Mutant p53 in Ovarian Carcinoma

The EphA2 tyrosine kinase receptor is frequently overexpressed in ovarian cancer and this feature is predictive of poor clinical outcome. Pre-clinical investigation has also linked EphA2 with p53. In our present study, we examined EphA2 and p53 status (both expression and full-length mutation status) in 6 ovarian cell lines and 79 human ovarian cancers to determine potential associations. EphA2 was overexpressed in 80% of ovarian cancer cell lines and in 75% of clinical specimens. In particular, high levels of EphA2 occurred in 91% of tumors with p53 null mutations compared to 68% in tumors with wild-type or missense mutations (p=0.027). EphA2 expression did not relate to critical versus non-critical site missense p53 mutations or the location of mutations on specific p53 exons. We also demonstrated that while EphA2 and p53 can provide independent information regarding clinical status, the combination of EphA2 and p53 status can predict poor clinical outcome. In particular, the combination of EphA2 overexpression and p53 null status was associated with decreased overall patient survival and related to increased incidence of ascites and distant metastasis. Taken together, these data indicate a complex relationship between EphA2 and p53 that appears to regulate EphA2 expression and clinical outcome.