Alpa M. Nick

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.

Paraneoplastic Thrombocytosis in Ovarian Cancer

BACKGROUND The mechanisms of paraneoplastic thrombocytosis in ovarian cancer and the role that platelets play in abetting cancer growth are unclear. METHODS We analyzed clinical data on 619 patients with epithelial ovarian cancer to test associations between platelet counts and disease outcome. Human samples and mouse models of epithelial ovarian cancer were used to explore the underlying mechanisms of paraneoplastic thrombocytosis. The effects of platelets on tumor growth and angiogenesis were ascertained. RESULTS Thrombocytosis was significantly associated with advanced disease and shortened survival. Plasma levels of thrombopoietin and interleukin-6 were significantly elevated in patients who had thrombocytosis as compared with those who did not. In mouse models, increased hepatic thrombopoietin synthesis in response to tumor-derived interleukin-6 was an underlying mechanism of paraneoplastic thrombocytosis. Tumor-derived interleukin-6 and hepatic thrombopoietin were also linked to thrombocytosis in patients. Silencing thrombopoietin and interleukin-6 abrogated thrombocytosis in tumor-bearing mice. Anti-interleukin-6 antibody treatment significantly reduced platelet counts in tumor-bearing mice and in patients with epithelial ovarian cancer. In addition, neutralizing interleukin-6 significantly enhanced the therapeutic efficacy of paclitaxel in mouse models of epithelial ovarian cancer. The use of an antiplatelet antibody to halve platelet counts in tumor-bearing mice significantly reduced tumor growth and angiogenesis. CONCLUSIONS These findings support the existence of a paracrine circuit wherein increased production of thrombopoietic cytokines in tumor and host tissue leads to paraneoplastic thrombocytosis, which fuels tumor growth. We speculate that countering paraneoplastic thrombocytosis either directly or indirectly by targeting these cytokines may have therapeutic potential. (Funded by the National Cancer Institute and others.).

Sustained Small Interfering RNA Delivery by Mesoporous Silicon Particles

RNA interference (RNAi) is a powerful approach for silencing genes associated with a variety of pathologic conditions; however, in vivo RNAi delivery has remained a major challenge due to lack of safe, efficient, and sustained systemic delivery. Here, we report on a novel approach to overcome these limitations using a multistage vector composed of mesoporous silicon particles (stage 1 microparticles, S1MP) loaded with neutral nanoliposomes (dioleoyl phosphatidylcholine, DOPC) containing small interfering RNA (siRNA) targeted against the EphA2 oncoprotein, which is overexpressed in most cancers, including ovarian. Our delivery methods resulted in sustained EphA2 gene silencing for at least 3 weeks in two independent orthotopic mouse models of ovarian cancer following a single i.v. administration of S1MP loaded with EphA2-siRNA-DOPC. Furthermore, a single administration of S1MP loaded with-EphA2-siRNA-DOPC substantially reduced tumor burden, angiogenesis, and cell proliferation compared with a noncoding control siRNA alone (SKOV3ip1, 54%; HeyA8, 57%), with no significant changes in serum chemistries or in proinflammatory cytokines. In summary, we have provided the first in vivo therapeutic validation of a novel, multistage siRNA delivery system for sustained gene silencing with broad applicability to pathologies beyond ovarian neoplasms.

Position-related injury is uncommon in robotic gynecologic surgery ☆ ☆☆

OBJECTIVE To assess the rate and risk factors for position-related injury in robotic gynecologic surgery. METHODS A prospective database from 12/2006 to 1/2014 of all planned robotic gynecologic procedures was retrospectively reviewed for patients who experienced neurologic injury, musculoskeletal injury, or vascular compromise related to patient positioning in the operating room. Analysis was performed to determine risk-factors and incidence for position-related injury. RESULTS Of the 831 patients who underwent robotic surgery during the study time period, only 7 (0.8%) experienced positioning-related injury. The injuries included minor head contusions (n=3), two lower extremity neuropathies (n=2), brachial plexus injury (n=1) and one large subcutaneous ecchymosis on the left flank and thigh (n=1). There were no long term sequelae from the positioning-related injuries. The only statistically significant risk factor for positioning-related injury was prior abdominal surgery (P=0.05). There were no significant associations between position-related injuries and operative time (P=0.232), body mass index (P=0.847), age (P=0.152), smoking history (P=0.161), or medical comorbidities (P=0.229-0.999). CONCLUSIONS The incidence of position-related injury among women undergoing robotic surgery was extremely low (0.8%). Due to the low incidence we were unable to identify modifiable risk factors for position-related injury following robotic surgery. A standardized, team-oriented approach may significantly decrease position-related injuries following robotic gynecologic surgery.

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.

Targeted Gene Silencing Using RGD-Labeled Chitosan Nanoparticles

Purpose: This study aimed to develop an Arg-Gly-Asp (RGD) peptide-labeled chitosan nanoparticle (RGD-CH-NP) as a novel tumor targeted delivery system for short interfering RNA (siRNA). Experimental Design: RGD peptide conjugated with chitosan by thiolation reaction was confirmed by proton-NMR (H-NMR). Binding of RGD-CH-NP with ανβ3 integrin was examined by flow cytometry and fluorescence microscopy. Antitumor efficacy was examined in orthotopic mouse models of ovarian carcinoma. Results: We show that RGD-CH-NP loaded with siRNA significantly increased selective intratumoral delivery in orthotopic animal models of ovarian cancer. In addition, we show targeted silencing of multiple growth-promoting genes (POSTN, FAK, and PLXDC1) along with therapeutic efficacy in the SKOV3ip1, HeyA8, and A2780 models using siRNA incorporated into RGD-CH-NP (siRNA/RGD-CH-NP). Furthermore, we show in vivo tumor vascular targeting using RGD-CH-NP by delivering PLXDC1-targeted siRNA into the ανβ3 integrin–positive tumor endothelial cells in the A2780 tumor-bearing mice. This approach resulted in significant inhibition of tumor growth compared with controls. Conclusions: This study shows that RGD-CH-NP is a novel and highly selective delivery system for siRNA with the potential for broad applications in human disease. Clin Cancer Res; 16(15); 3910–22. ©2010 AACR.

Targeting Aldehyde Dehydrogenase Cancer Stem Cells in Ovarian Cancer

Aldehyde dehydrogenase-1A1 (ALDH1A1) expression characterizes a subpopulation of cells with tumor-initiating or cancer stem cell properties in several malignancies. Our goal was to characterize the phenotype of ALDH1A1-positive ovarian cancer cells and examine the biological effects of ALDH1A1 gene silencing. In our analysis of multiple ovarian cancer cell lines, we found that ALDH1A1 expression and activity was significantly higher in taxane- and platinum-resistant cell lines. In patient samples, 72.9% of ovarian cancers had ALDH1A1 expression in which the percentage of ALDH1A1-positive cells correlated negatively with progression-free survival (6.05 vs. 13.81 months; P < 0.035). Subpopulations of A2780cp20 cells with ALDH1A1 activity were isolated for orthotopic tumor–initiating studies, where tumorigenicity was approximately 50-fold higher with ALDH1A1-positive cells. Interestingly, tumors derived from ALDH1A1-positive cells gave rise to both ALDH1A1-positive and ALDH1A1-negative populations, but ALDH1A1-negative cells could not generate ALDH1A1-positive cells. In an in vivo orthotopic mouse model of ovarian cancer, ALDH1A1 silencing using nanoliposomal siRNA sensitized both taxane- and platinum-resistant cell lines to chemotherapy, significantly reducing tumor growth in mice compared with chemotherapy alone (a 74%–90% reduction; P < 0.015). These data show that the ALDH1A1 subpopulation is associated with chemoresistance and outcome in ovarian cancer patients, and targeting ALDH1A1 sensitizes resistant cells to chemotherapy. ALDH1A1-positive cells have enhanced, but not absolute, tumorigenicity but do have differentiation capacity lacking in ALDH1A1-negative cells. This enzyme may be important for identification and targeting of chemoresistant cell populations in ovarian cancer. Mol Cancer Ther; 9(12); 3186–99. ©2010 AACR.

Therapeutic Efficacy of a Novel Focal Adhesion Kinase Inhibitor TAE226 in Ovarian Carcinoma

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.

Adrenergic modulation of focal adhesion kinase protects human ovarian cancer cells from anoikis

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.

Surgical stress promotes tumor growth in ovarian carcinoma.

Purpose: Surgical stress has been suggested to facilitate the growth of preexisting micrometastases as well as small residual tumor postoperatively. The purpose of this study was to examine the effects of surgical stress on ovarian cancer growth and to determine underlying mechanisms responsible for increased growth. Experimental Design: To mimic the effects of surgery, we did a laparotomy or mastectomy under isoflurane inhalation on athymic nude mice 4 days after i.p. tumor cell injection. Propranolol infusion via Alzet pumps was used to block the influence of sympathetic nervous system activation by surgical stress. Results: In both HeyA8 and SKOV3ip1 models, the mice in the laparotomy and mastectomy groups had significantly greater tumor weight (P < 0.05) and nodules (P < 0.05) compared with anesthesia only controls. There was no increase in tumor weight following surgery in the β-adrenergic receptor–negative RMG-II model. Propranolol completely blocked the effects of surgical stress on tumor growth, indicating a critical role for β-adrenergic receptor signaling in mediating the effects of surgical stress on tumor growth. In the HeyA8 and SKOV3ip1 models, surgery significantly increased microvessel density (CD31) and vascular endothelial growth factor expression, which were blocked by propranolol treatment. Conclusion: These results indicate that surgical stress could enhance tumor growth and angiogenesis, and β-blockade might be effective in preventing such effects.