Chronic stress promotes tumor growth and angiogenesis in a mouse model of ovarian carcinoma.

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 beta(2) 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 beta-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.

Phase 1-2 study of docetaxel plus aflibercept in patients with recurrent ovarian, primary peritoneal, or fallopian tube cancer.

BACKGROUND: Biologically targeted therapies have been postulated as a viable strategy to improve outcomes for women with ovarian cancer. We assessed the safety, tolerance, pharmacokinetics, relevant circulating and image-derived biomarkers, and clinical activity of combination aflibercept and docetaxel in this population. METHODS: For the phase 1 (pharmacokinetic) study, eligible patients had measurable, recurrent or persistent epithelial ovarian, primary peritoneal, or fallopian tube carcinoma with a maximum of two prior chemotherapy regimens. Aflibercept was administered intravenously over three dose levels (2, 4, or 6 mg/kg; one dose every 21 days) to identify the maximum tolerated dose for the phase 2 study. Pharmacokinetics were assessed and dynamic imaging was done during a lead-in phase with single-agent aflibercept (cycle 0) and during combination therapy with intravenous docetaxel (75 mg/m(2)). Eligibility for the phase 2 study was the same as for phase 1. Patients were enrolled in a two-stage design and given aflibercept 6 mg/kg intravenously and docetaxel 75 mg/m(2) intravenously, every 3 weeks. The primary endpoint was objective response rate (ORR) as assessed by Response Evaluation Criteria in Solid Tumors version 1.0. The trial has completed enrolment and all patients are now off study. The trial is registered at ClinicalTrials.gov, number NCT00436501. FINDINGS: From the phase 1 study, the recommended phase 2 doses of aflibercept and docetaxel were found to be 6 mg/kg and 75 mg/m(2), respectively. Log-linear pharmacokinetics (for unbound aflibercept) were observed for the three dose levels. No dose-limiting toxicities were noted. 46 evaluable patients were enrolled in the phase 2 trial; 33 were platinum resistant (15 refractory) and 13 were platinum sensitive. The confirmed ORR was 54% (25 of 46; 11 patients had a complete response and 14 had a partial response). Grade 3-4 toxicities observed in more than two patients (5%) were: neutropenia in 37 patients (80%); leucopenia in 25 patients (54%); fatigue in 23 patients (50%); dyspnoea in ten patients (22%); and stomatitis in three patients (7%). Adverse events specifically associated with aflibercept were grade 1-2 hypertension in five patients (11%), and grade 2 proteinuria in one patient (2%). INTERPRETATION: Combination aflibercept plus docetaxel can be safely administered at the dose and schedule reported here, and is associated with substantial antitumour activity. These findings suggest that further clinical development of this combination in ovarian cancer is warranted. FUNDING: US National Cancer Institute, US Department of Defense, Sanofi-Aventis, Gynecologic Cancer Foundation, Marcus Foundation, and the Commonwealth Foundation.

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.

Functional significance of VEGFR-2 on ovarian cancer cells.

Vascular endothelial growth factor receptor (VEGFR) has recently been discovered on ovarian cancer cells, but its functional significance is unknown and is the focus of this study. By protein analysis, A2780-par and HeyA8 ovarian cancer cell lines expressed VEGFR-1 and HeyA8 A2774, and SKOV3ip1 expressed VEGFR-2. By in situ hybridization (ISH), 85% of human ovarian cancer specimens showed moderate to high VEGFR-2 expression, whereas only 15% showed moderate to high VEGFR-1 expression. By immunofluorescence, little or no VEGFR-2 was detected in normal ovarian surface epithelial cells, whereas expression was detected in 75% of invasive ovarian cancer specimens. To differentiate between the effects of tumor versus host expression of VEGFR, nude mice were injected with SKOV3ip1 cells and treated with either human VEGFR-2 specific antibody (1121B), murine VEGFR-2 specific antibody (DC101) or the combination. Treatment with 1121B reduced SKOV3ip1 cell migration by 68% (p < 0.01) and invasion by 72% (p < 0.01), but exposure to VEGFR-1 antibody had no effect. Treatment with 1121B effectively blocked VEGF-induced phosphorylation of p130Cas. In vivo treatment with either DC101 or 1121B significantly reduced tumor growth alone and in combination in the SKOV3ip1 and A2774 models. Decreased tumor burden after treatment with DC101 or 1121B correlated with increased tumor cell apoptosis, decreased proliferative index, and decreased microvessel density. These effects were significantly greater in the combination group (p < 0.001). We show functionally active VEGFR-2 is present on most ovarian cancer cells. The observed anti-tumor activity of VEGF-targeted therapies may be mediated by both anti-angiogenic and direct anti-tumor effects.

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), beta 2 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 beta 2 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 < or = 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.

Targeting aurora kinase with MK-0457 inhibits ovarian cancer growth.

PURPOSE: The Aurora kinase family plays pivotal roles in mitotic integrity and cell cycle. We sought to determine the effects of inhibiting Aurora kinase on ovarian cancer growth in an orthotopic mouse model using a small molecule pan-Aurora kinase inhibitor, MK-0457. EXPERIMENTAL DESIGN: We examined cell cycle regulatory effects and ascertained the therapeutic efficacy of Aurora kinase inhibition both alone and combined with docetaxel using both in vitro and in vivo ovarian cancer models. RESULTS: In vitro cytotoxicity assays with HeyA8 and SKOV3ip1 cells revealed >10-fold greater docetaxel cytotoxicity in combination with MK-0457. After in vivo dose kinetics were determined using phospho-histone H3 status, therapy experiments with the chemosensitive HeyA8 and SKOV3ip1 as well as the chemoresistant HeyA8-MDR and A2780-CP20 models showed that Aurora kinase inhibition alone significantly reduced tumor burden compared with controls (P values<0.01). Combination treatment with docetaxel resulted in significantly improved reduction in tumor growth beyond that afforded by docetaxel alone (P

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.

Metronomic chemotherapy enhances the efficacy of antivascular therapy in ovarian cancer.

Metronomic chemotherapy is the frequent administration of low doses of chemotherapeutic agents targeting tumor-associated endothelial cells. We examined the efficacy of metronomic taxanes alone and in combination with AEE788-a dual epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) inhibitor-in an orthotopic mouse model of ovarian cancer. Growth-modulating effects of metronomic and maximum tolerated dose (MTD) regimens on overall survival were tested in vivo using both chemotherapy-sensitive (HeyA8 and SKOV3ip1) and chemotherapy-resistant (HeyA8-MDR) models. Treated tumors were stained for microvessel density (CD31), proliferation index (proliferating cell nuclear antigen), and apoptosis (terminal deoxyribonucleotide transferase-mediated nick-end labeling). The cytotoxic effects of MTD and metronomic dosing were tested with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Effects of metronomic regimens on circulating endothelial precursors (CEP) and tumor-specific cell-free DNA levels were assessed. In vivo, metronomic docetaxel resulted in significant reduction of tumor growth in the taxane-sensitive cell lines, whereas metronomic docetaxel plus AEE788 had an additive effect resulting in significant prolongation in survival. Combination therapy was effective even in the taxane-resistant model. Metronomic chemotherapy alone and combined with AEE788 resulted in a decrease in the proliferative index and microvessel density of treated tumors, whereas combination therapy increased the apoptotic index (P < 0.001). In vitro, metronomic taxanes caused endothelial cell toxicity at 10- to 100-fold lower concentrations compared with MTD dosing. Metronomic regimens inhibited mobilization of CEPs (P < 0.05) and led to a decrease in cell-free DNA levels (P < 0.05). Our results suggest that metronomic taxane chemotherapy with dual EGFR and VEGFR inhibition is highly efficacious and should be considered for future clinical trials.

Antitumor and antivascular effects of AVE8062 in ovarian carcinoma.

The purpose of this study was to examine the therapeutic efficacy and underlying mechanisms of action of a vascular-disrupting agent, AVE8062, and to determine its effects on tumor metabolic activity. The in vitro and in vivo effects of AVE8062 alone and in combination with docetaxel were tested in chemotherapy-sensitive and chemotherapy-resistant ovarian cancer models. Tumors were analyzed for necrosis, microvessel density, endothelial cell apoptosis, and proliferation following treatment. The effect of AVE8062 on tumor regression and metabolic activity was examined by magnetic resonance (MR) or by [18F]fluorodeoxyglucose ([18F]FDG) uptake by positron emission tomography (PET) with MR imaging, respectively. AVE8062 monotherapy was effective in inhibiting tumor growth in all models (range 43-51% versus control; P < 0.05). Combination therapy was even more effective in inhibiting tumor growth (range 76-90% compared with controls, P < 0.01). AVE8062 in combination with chemotherapy significantly prolonged survival in HeyA8-injected mice (P < 0.001) compared with other groups. AVE8062-based therapy resulted in rapid development of central tumor necrosis, decreased microvessel density, decreased proliferation, and induction of apoptosis of tumor-associated endothelial cells. MR imaging showed regression of established HeyA8 ovarian tumors and [18F]FDG PET with MR showed rapid decrease in metabolic activity after AVE8062 therapy. Combination of AVE8062 plus docetaxel results in potent inhibition of ovarian cancer growth. These results suggest that AVE8062 may be useful as a clinical therapeutic approach for ovarian cancer patients and that functional [18F]FDG PET imaging may predict clinical response before an anatomic reduction in tumor size.

Relaxin promotes prostate cancer progression.

PURPOSE: To understand the role of relaxin peptide in prostate cancer, we analyzed the expression of relaxin and its receptor in human prostate cancer samples, the effects of relaxin signaling on cancer cell phenotype in vitro, and the effects of increased serum relaxin concentrations on cancer progression in vivo. EXPERIMENTAL DESIGN: The relaxin and its receptor leucine-rich repeat containing G protein-coupled receptor 7 (LGR7) expression were studied by quantitative reverse transcription-PCR (11 benign and 44 cancer tissue samples) and by relaxin immunohistochemistry using tissue microarrays containing 10 normal and 69 cancer samples. The effects of relaxin treatment and endogenous relaxin/LGR7 suppression via short interfering RNA in PC-3 and LNCaP cells were analyzed in vitro. The effect of transgenic relaxin overexpression [Tg(Rln1)] on cancer growth and survival was evaluated in autochthonous transgenic adenocarcinoma of the mouse prostate (TRAMP). RESULTS: The relaxin mRNA expression was significantly higher in recurrent prostate cancer samples. In tissue microarrays of the 10 normal tissues, 8 had low staining in epithelial cells, whereas only 1 of 9 high-grade prostatic intraepithelial neoplasia lesions had low expression (P = 0.005) and only 29 of 65 cancers had low expression (P = 0.047). Stimulation with relaxin increased cell proliferation, invasiveness, and adhesion in vitro. The suppression of relaxin/LGR7 via short interfering RNAs decreased cell invasiveness by 90% to 95% and growth by 10% to 25% and increased cell apoptosis 0.6 to 2.2 times. The Tg(Rln1) TRAMP males had shorter median survival time, associated with the decreased apoptosis of tumor cells, compared with non-Tg(Rln1) TRAMP animals. CONCLUSIONS: Relaxin signaling plays a role in prostate cancer progression.

Curcumin inhibits tumor growth and angiogenesis in ovarian carcinoma by targeting the nuclear factor-kappaB pathway.

PURPOSE: Curcumin, a component of turmeric, has been shown to suppress inflammation and angiogenesis largely by inhibiting the transcription factor nuclear factor-kappaB (NF-kappaB). This study evaluates the effects of curcumin on ovarian cancer growth using an orthotopic murine model of ovarian cancer. EXPERIMENTAL DESIGN: In vitro and in vivo experiments of curcumin with and without docetaxel were done using human ovarian cancer cell lines SKOV3ip1, HeyA8, and HeyA8-MDR in athymic mice. NF-kappaB modulation was ascertained using electrophoretic mobility shift assay. Evaluation of angiogenic cytokines, cellular proliferation (proliferating cell nuclear antigen), angiogenesis (CD31), and apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) was done using immunohistochemical analyses. RESULTS: Curcumin inhibited inducible NF-kappaB activation and suppressed proliferation in vitro. In vivo dose-finding experiments revealed that 500 mg/kg orally was the optimal dose needed to suppress NF-kappaB and signal transducers and activators of transcription 3 activation and decrease angiogenic cytokine expression. In the SKOV3ip1 and HeyA8 in vivo models, curcumin alone resulted in 49% (P = 0.08) and 55% (P = 0.01) reductions in mean tumor growth compared with controls, whereas when combined with docetaxel elicited 96% (P < 0.001) and 77% reductions in mean tumor growth compared with controls. In mice with multidrug-resistant HeyA8-MDR tumors, treatment with curcumin alone and combined with docetaxel resulted in significant 47% and 58% reductions in tumor growth, respectively (P = 0.05). In SKOV3ip1 and HeyA8 tumors, curcumin alone and with docetaxel decreased both proliferation (P < 0.001) and microvessel density (P < 0.001) and increased tumor cell apoptosis (P < 0.05). CONCLUSIONS: Based on significant efficacy in preclinical models, curcumin-based therapies may be attractive in patients with ovarian carcinoma.

Dual targeting of endothelial cells and pericytes in antivascular therapy for ovarian carcinoma.

PURPOSE: Pericytes are known to provide a survival advantage for endothelial cells. We hypothesize that strategies aimed at dual targeting of tumor-associated endothelial cells and pericytes will be highly efficacious. EXPERIMENTAL DESIGN: Paclitaxel-sensitive (HeyA8 and SKOV3ip1) or paclitaxel-resistant (HeyA8-MDR) orthotopic tumors in mice were examined for therapeutic efficacy by targeting the endothelial cells (using a vascular endothelial growth factor receptor inhibitor, AEE788) and pericytes (using STI571) alone or in combination. Additional therapy and survival studies in combination with paclitaxel were also done. Following therapy, tumors were examined for endothelial cell apoptosis, pericyte coverage, microvessel density, and proliferation. RESULTS: AEE788 inhibited tumor growth by 45% and 59% in the HeyA8 and SKOV3ip1 models, respectively, whereas STI571 alone was not effective. AEE788 plus STI571 resulted in 69% to 84% inhibition of tumor growth in both models. Moreover, combination of these agents with paclitaxel was even more effective, resulting in up to 98% inhibition of tumor growth. The triple combination was even effective in the HeyA8-MDR model. Remarkably, this triple combination also resulted in improved survival compared with all other groups (P<0.001) and caused regression of formed tumors. Pericyte coverage was significantly decreased in the STI571 treatment groups, and microvessel density was significantly reduced in the AEE788 treatment groups. AEE788 induced endothelial cell apoptosis, which was further enhanced by the addition of STI571. CONCLUSIONS: Strategies targeting both endothelial cells and pericytes are highly effective for in vivo treatment of ovarian carcinoma. This antiangiogenic effect may be partially due to decreased pericyte coverage, thus increasing the sensitivity of tumor vasculature to therapy. These encouraging data support the development of clinical trials based on this strategy.

Clinical and biological significance of vascular endothelial growth factor in endometrial cancer.

PURPOSE: 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. EXPERIMENTAL DESIGN: 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. RESULTS: 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). CONCLUSIONS: 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.

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.

Stress hormone-mediated invasion of ovarian cancer cells.

PURPOSE: There is growing evidence that stress and other behavioral factors may affect cancer progression and patient survival. The underlying mechanisms for this association are poorly understood. The purpose of this study is to determine the effects of stress-associated hormones norepinephrine, epinephrine, and cortisol on the invasive potential of ovarian cancer cells. EXPERIMENTAL DESIGN: The ovarian cancer cells EG, SKOV3, and 222 were exposed to increasing levels of either norepinephrine, epinephrine, or cortisol, and the in vitro invasive potential was determined using the membrane invasion culture system. Additionally, the effects of these stress hormones on matrix metalloproteinase-2 (MMP-2) and MMP-9 were determined by ELISA. The effects of the beta-adrenergic agonist isoproterenol on in vivo tumor growth were determined using nude mice. RESULTS: Stress levels of norepinephrine increased the in vitro invasiveness of ovarian cancer cells by 89% to 198%. Epinephrine also induced significant increases in invasion in all three cell lines ranging from 64% to 76%. Cortisol did not significantly affect invasiveness of the EG and 222 cell lines but increased invasion in the SKOV3 cell line (P = 0.01). We have previously shown that ovarian cancer cells express beta-adrenergic receptors. The beta-adrenergic antagonist propanolol (1 mumol/L) completely blocked the norepinephrine-induced increase in invasiveness. Norepinephrine also increased tumor cell expression of MMP-2 (P = 0.02 for both SKOV3 and EG cells) and MMP-9 (P = 0.01 and 0.04, respectively), and pharmacologic blockade of MMPs abrogated the effects of norepinephrine on tumor cell invasive potential. Isoproterenol treatment resulted in a significant increase in tumor volume and infiltration in the SKOV3ip1 in vivo model, which was blocked by propranolol. CONCLUSIONS: These findings provide direct experimental evidence that stress hormones can enhance the invasive potential of ovarian cancer cells. These effects are most likely mediated by stimulation of MMPs.

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 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. CONCLUSIONS: Taken together, these findings suggest that FAK siRNA-DOPC plus docetaxel or platinum might be a novel therapeutic approach against ovarian cancer.

Endocrine effects of relaxin overexpression in mice.

Relaxin is a small peptide hormone with a variety of biological functions. To investigate the systemic endocrine effects of relaxin, we produced mice with transgenic overexpression of the Rln1 gene, Tg(Rln1), driven by rat insulin 2 promoter. The expression of relaxin was detected in the pancreas of the transgenic animals. An analysis of the sera from the transgenic animals revealed at least 20-fold elevation of the level of bioactive relaxin. Transgenic animals had normal viability and fertility in both sexes. Transgenic overexpression of Rln1 did not rescue the undescended testis phenotype in Insl3-deficient males, suggesting that in vivo relaxin does not interact with the insulin-like 3 factor receptor, leucine-rich repeats-containing G protein-coupled receptor 8, Lgr8. Phenotypically, the excess of relaxin resulted in hypertrophic nipple development in virgin female mice. Deletion of the relaxin receptor, leucine-rich repeats-containing G protein-coupled receptor 7, Lgr7, in Tg(Rln1) animals abrogated the development of enlarged nipples in females, indicating that relaxin exerts its effect through Lgr7 alone. The levels of previously defined targets of relaxin signaling, such as matrix metalloproteinases 2 and 9, vascular endothelial growth factor, or nitric oxide, were similar in the sera of the transgenic and wild-type mice. However, the total plasma protein concentration in male Tg(Rln1) mice was lower than that in control animals. The livers of male Tg(Rln1) mice exhibited significantly higher hydroxyproline content, indicative of increased collagen deposition. Our results indicate that relaxin overexpression causes gender-specific changes in liver collagen metabolism.

The role of relaxin in endometrial cancer.

Relaxin (RLN) is a naturally occurring hormone that is known to modulate connective tissue remodeling in the uterus and cervix. Our goal was to investigate the role of RLN in endometrial cancer. RLN expression was evaluated using immunohistochemistry in 57 samples of invasive endometrial carcinoma (EC) and ten benign endometrial tissues. 67% of high-stage (III/IV) tumors demonstrated strong RLN expression compared to 37% of low-stage (I/II) cases. Strong RLN expression associated significantly with high-grade and depth of myometrial invasion. Notably, strong RLN expression was associated with a significantly shorter overall survival (p < 0.005) compared to weak or moderate expression. Using RT-PCR, the expression of RLN and its receptor (LGR7) was detected in EC cell lines (HEC-1B and KLE); in addition, LGR7 was expressed in 86% of 15 primary EC tissue samples. Exogenous RLN stimulation caused a significant increase in migration and invasion in both cell lines, but did not stimulate proliferation in vitro. Addition of the MMP inhibitor, FN439 abolished the stimulatory effect of RLN on invasion in both HEC-1B and KLE cells. RLN stimulation caused a significant increase in levels of activated MMP-2 in KLE cells and activated MMP-9 in HEC-1B cells compared to unstimulated cells. Inhibition of endogenous RLN signaling via siRNA targeted to LGR7 caused a significant reduction of EC cell invasiveness. Our results indicate that RLN overexpression is significantly asso- ciated with aggressive features such as high-grade and deep myometrial invasion. We provide the first evidence that overexpression of RLN is associated with poor clinical outcome in women with EC. RLN stimulation enhances the invasive potential of endometrial cancer cells by upregulating MMPs. In turn, downregulation of endogenous RLN signaling decreases invasiveness of endometrial cancer cells. These novel findings may have therapeutic implications in the management of patients with endometrial carcinoma.

Circulating cell-free DNA: a novel biomarker for response to therapy in ovarian carcinoma.

INTRODUCTION: Cell-free DNA (CFDNA) is a reflection of both normal and tumor-derived DNA released into the circulation through cellular necrosis and apoptosis. We sought to determine whether tumor-specific plasma DNA could be used as a biomarker for tumor burden and response to therapy in an orthotopic ovarian cancer model. METHODS: Female nude mice injected intraperitoneally with HeyA8 ovarian cancer cells were treated with either docetaxel alone or in combination with anti-angiogenic agents (AEE788-dual VEGFR and EGFR antagonist or EA5-monoclonal antibody against ephrin A2). Following DNA extraction from plasma, quantification of tumor-specific DNA was performed by real-time PCR using human specific beta-actin primers. The number of genome equivalents (GE/ml) were determined from a standard curve. Apoptosis was assessed by TUNEL staining of treated tumors. RESULTS: The levels of tumor-specific DNA in plasma increased progressively with increasing tumor burden (R2=0.8, p<0.01). Additionally, tumor-specific plasma DNA levels varied following treatment with chemotherapy. In mice with established tumors (19 days following tumor injection), tumor-specific plasma DNA levels increased by 63% at 24 hours following a single dose of docetaxel (15 mg/kg), and then declined to 20% below baseline at 72 hours and were 83% lower than baseline 10 days following therapy. In addition, docetaxel treatment resulted in a significant increase in the apoptotic index at 24 hours (p<0.01). Moreover, in two separate therapy experiments using a combination of cytotoxic chemotherapy with anti-angiogenic agents, tumor-specific plasma DNA levels were significantly higher in mice treated with vehicle compared to the treatment groups. The correlation between tumor weight and tumor-specific DNA in these experiments was 0.71-0.76 (p<0.01). CONCLUSIONS: Our results indicate that tumor-specific CFDNA levels correlate with increasing tumor burden and decline following therapy. Thus, tumor-specific DNA may be a useful surrogate biomarker of therapeutic response and should be evaluated in future clinical trials.

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. Preclinical 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.