A. Ziebarth

Stem Cell Pathways Contribute to Clinical Chemoresistance in Ovarian Cancer

Purpose: Within heterogeneous tumors, subpopulations often labeled cancer stem cells (CSC) have been identified that have enhanced tumorigenicity and chemoresistance in ex vivo models. However, whether these populations are more capable of surviving chemotherapy in de novo tumors is unknown. Experimental Design: We examined 45 matched primary/recurrent tumor pairs of high-grade ovarian adenocarcinomas for expression of CSC markers ALDH1A1, CD44, and CD133 using immunohistochemistry. Tumors collected immediately after completion of primary therapy were then laser capture microdissected and subjected to a quantitative PCR array examining stem cell biology pathways (Hedgehog, Notch, TGF-β, and Wnt). Select genes of interest were validated as important targets using siRNA-mediated downregulation. Results: Primary samples were composed of low densities of ALDH1A1, CD44, and CD133. Tumors collected immediately after primary therapy were more densely composed of each marker, whereas samples collected at first recurrence, before initiating secondary therapy, were composed of similar percentages of each marker as their primary tumor. In tumors collected from recurrent platinum-resistant patients, only CD133 was significantly increased. Of stem cell pathway members examined, 14% were significantly overexpressed in recurrent compared with matched primary tumors. Knockdown of genes of interest, including endoglin/CD105 and the hedgehog mediators Gli1 and Gli2, led to decreased ovarian cancer cell viability, with Gli2 showing a novel contribution to cisplatin resistance. Conclusions: These data indicate that ovarian tumors are enriched with CSCs and stem cell pathway mediators, especially at the completion of primary therapy. This suggests that stem cell subpopulations contribute to tumor chemoresistance and ultimately recurrent disease. Clin Cancer Res; 18(3); 869–81. ©2011 AACR.

Smoothened Antagonists Reverse Taxane Resistance in Ovarian Cancer

The hedgehog pathway has been implicated in the formation and maintenance of a variety of malignancies, including ovarian cancer; however, it is unknown whether hedgehog signaling is involved in ovarian cancer chemoresistance. The goal of this study was to determine the effects of antagonizing the hedgehog receptor, Smoothened (Smo), on chemotherapy response in ovarian cancer. Expression of hedgehog pathway members was assessed in three pairs of parental and chemotherapy-resistant ovarian cancer cell lines (A2780ip2/A2780cp20, SKOV3ip1/SKOV3TRip2, HeyA8/HeyA8MDR) using quantitative PCR and Western blot analysis. Cell lines were exposed to increasing concentrations of two different Smo antagonists (cyclopamine, LDE225) alone and in combination with carboplatin or paclitaxel. Selective knockdown of Smo, Gli1, or Gli2 was achieved using siRNA constructs. Cell viability was assessed by MTT assay. A2780cp20 and SKOV3TRip2 orthotopic xenografts were treated with vehicle, LDE225, paclitaxel, or combination therapy. Chemoresistant cell lines showed higher expression (>2-fold, P < 0.05) of hedgehog signaling components compared with their respective parental lines. Smo antagonists sensitized chemotherapy-resistant cell lines to paclitaxel, but not to carboplatin. LDE225 treatment also increased sensitivity of ALDH-positive cells to paclitaxel. A2780cp20 and SKOV3TRip2 xenografts treated with combined LDE225 and paclitaxel had significantly less tumor burden than those treated with vehicle or either agent alone. Increased taxane sensitivity seems to be mediated by a decrease in P-glycoprotein (MDR1) expression. Selective knockdown of Smo, Gli1, or Gli2 all increased taxane sensitivity. Smo antagonists reverse taxane resistance in chemoresistant ovarian cancer models, suggesting combined anti-hedgehog and chemotherapies could provide a useful therapeutic strategy for ovarian cancer. Mol Cancer Ther; 11(7); 1587–97. ©2012 AACR.

Endoglin (CD105) Contributes to Platinum Resistance and Is A Target for Tumor-Specific Therapy in Epithelial Ovarian Cancer

Purpose: Endoglin (CD105) is a membranous protein overexpressed in tumor-associated endothelial cells, chemoresistant populations of ovarian cancer cells, and potentially stem cells. Our objective was to evaluate the effects and mechanisms of targeting endoglin in ovarian cancer. Experimental Design: Global and membranous endoglin expression was evaluated in multiple ovarian cancer lines. In vitro, the effects of siRNA-mediated endoglin knockdown with and without chemotherapy were evaluated by MTT assay, cell-cycle analysis, alkaline comet assay, γ-H2AX foci formation, and quantitative PCR. In an orthotopic mouse model, endoglin was targeted with chitosan-encapsulated siRNA with and without carboplatin. Results: Endoglin expression was surprisingly predominantly cytoplasmic, with a small population of surface-positive cells. Endoglin inhibition decreased cell viability, increased apoptosis, induced double-stranded DNA damage, and increased cisplatin sensitivity. Targeting endoglin downregulates expression of numerous DNA repair genes, including BARD1, H2AFX, NBN, NTHL1, and SIRT1. BARD1 was also associated with platinum resistance, and was induced by platinum exposure. In vivo, antiendoglin treatment decreased tumor weight in both ES2 and HeyA8MDR models when compared with control (35%–41% reduction, P < 0.05). Endoglin inhibition with carboplatin was associated with even greater inhibitory effect when compared with control (58%–62% reduction, P < 0.001). Conclusions: Endoglin downregulation promotes apoptosis, induces significant DNA damage through modulation of numerous DNA repair genes, and improves platinum sensitivity both in vivo and in vitro. Antiendoglin therapy would allow dual treatment of both tumor angiogenesis and a subset of aggressive tumor cells expressing endoglin and is being actively pursued as therapy in ovarian cancer. Clin Cancer Res; 19(1); 170–82. ©2012 AACR.

Molecular/genetic therapies in ovarian cancer: future opportunities and challenges.

Ovarian cancer is the most lethal gynecologic cancer. Traditional therapies have included surgical management and cytotoxic chemotherapy; however, treatment paradigms continue to shift from empiric cytotoxic chemotherapy to more individualized treatment. Recent research efforts have focused on determining and targeting the molecular biological mechanisms of ovarian cancer in an attempt to develop novel therapeutic modalities with the ultimate goal of improving outcome while limiting toxicity. This chapter reviews progress in the development of novel therapies directed at major pathways implicated in ovarian tumorigenesis including angiogenesis, PARP inhibition, signal transduction, antifolate therapies, death receptor-mediated therapies, histone deacetylase inhibition, immunotherapeutics, and oncolytics.

EDD enhances cell survival and cisplatin resistance and is a therapeutic target for epithelial ovarian cancer

The E3 ubiquitin ligase EDD is overexpressed in recurrent, platinum-resistant ovarian cancers, suggesting a role in tumor survival and/or platinum resistance. EDD knockdown by small interfering RNA (siRNA) induced apoptosis in A2780ip2, OVCAR5 and ES-2 ovarian cancer cells, correlating with loss of the prosurvival protein myeloid cell leukemia sequence 1 (Mcl-1) through a glycogen synthase kinase 3 beta-independent mechanism. SiRNA to EDD or Mcl-1 induced comparable levels of apoptosis in A2780ip2 and ES-2 cells. Stable overexpression of Mcl-1 protected cells from apoptosis following EDD knockdown, accompanied by a loss of endogenous, but not exogenous, Mcl-1 protein, suggesting that EDD regulated Mcl-1 synthesis. Indeed, EDD knockdown induced a 1.87-fold decrease in Mcl-1 messenger RNA and EDD transfection enhanced murine Mcl-1 promoter-driven luciferase expression 5-fold. To separate EDD survival and potential cisplatin resistance functions, we generated EDD shRNA stable cell lines that could survive initial EDD knockdown and showed that these cells were 4- to 21-fold more sensitive to cisplatin. Moreover, transient EDD overexpression in COS-7 cells was sufficient to promote cisplatin resistance 2.4-fold, dependent upon its E3 ligase activity. In vivo, mouse intraperitoneal ES-2 and A2780ip2 xenograft experiments showed that mice treated with EDD siRNA by nanoliposomal delivery [1,2-dioleoyl-sn-glycero-3-phophatidylcholine (DOPC)] and cisplatin had significantly less tumor burden than those treated with control siRNA/DOPC alone (ES-2, 77.9% reduction, P = 0.004; A2780ip2, 75.9% reduction, P = 0.042) or control siRNA/DOPC with cisplatin in ES-2 (64.4% reduction, P = 0.035), with a trend in A2780ip2 (60.3% reduction, P = 0.168). These results identify EDD as a dual regulator of cell survival and cisplatin resistance and suggest that EDD is a therapeutic target for ovarian cancer.

Completed versus aborted radical hysterectomy for node-positive stage IB cervical cancer in the modern era of chemoradiation therapy

OBJECTIVE Debate continues about optimal management of patients with node-positive stage I cervical cancer. Our objective was to determine if patient outcomes are affected by radical hysterectomy in the modern era of adjuvant chemoradiation. METHODS Cervical cancer patients diagnosed from 2000 to 2008 were identified. Demographics, therapy, clinicopathologic data, progression free survival (PFS), overall survival (OS), total radiation exposure, and grade 3-4 complications were analyzed by student t, Mann-Whitney, Fishers exact, Kaplan-Meier, and log rank tests. RESULTS This single-institution review evaluated forty-one of 334 (13.4%) patients scheduled to undergo radical hysterectomy that had gross nodal disease diagnosed intraoperatively. 15 underwent aborted radical hysterectomy following lymphadenectomy; the remaining 26 underwent radical hysterectomy and lymphadenectomy. Eleven patients undergoing radical hysterectomy underwent whole pelvic radiation therapy (WPRT) while 8 (30.7%) patients underwent WPRT and postoperative vaginal brachytherapy (BT) for local treatment secondary to close margins. All patients undergoing aborted radical hysterectomy underwent WPRT and BT. With mean follow-up of 42.3 months, there were no significant differences in urinary, gastrointestinal, or hematologic complications between groups. When comparing those undergoing radical hysterectomy to aborted radical hysterectomy, there were no significant differences in local recurrence (11.5% vs 26.7%, p=0.39) or distant recurrence (19.2% vs. 33.3%, p=0.45), PFS (74.9 months vs 46.8 months, p=0.106), or OS (91.8 months vs 69.4 months, p=0.886). CONCLUSIONS Treatment of patients with early stage cervical cancer and nodal metastasis may be tailored intraoperatively. Completion of radical hysterectomy and lymphadenectomy decreases radiation exposure without apparently compromising safety or outcome in the era of adjuvant chemoradiation.

Abstract 5475: Endoglin (CD105) is a target for ovarian cancer cell-specific therapy through induction of DNA damage.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Endoglin (ENG, CD105) is a protein markedly overexpressed in tumor-associated endothelial cells, and a target for anti-angiogenic therapy. Recently we have noted it to be overexpressed in chemoresistant ovarian cancer cells as well. Our objective was to evaluate the effects and mechanisms of targeting endoglin specifically in ovarian cancer cells. Methods: Endoglin expression was assessed in multiple ovarian cancer lines by Western blot, immunohistochemistry, and flow cytometry. Anti-endoglin siRNAs were used to downregulate expression in ES2 and HeyA8MDR. In vitro, the effects of endoglin-knockdown individually and with chemotherapy were evaluated by MTT assay, cell-cycle analysis, alkaline comet assay, γ-H2AX foci formation and qPCR array for mediators of DNA damage and repair. In an orthotopic murine model, mice inoculated with ES2 or HeyA8MDR cell lines were administered chitosan-encapsulated anti-ENG siRNA or control siRNA with and without carboplatin. PCNA, γH2AX, and 53BP1 IHC and the TUNEL assay were performed to examine biologic effects of endoglin knockdown. Results: Endoglin is expressed to varying degrees by multiple ovarian cancer cell lines. Expression was on the cell surface, consistent with its recognized role as a cofactor with TGF-beta receptor, in only 5% of cells, with most other cells having cytoplasmic expression. In ES2 and HeyA8MDR cell lines, endoglin inhibition decreased cell viability, increased apoptosis, induced double-stranded DNA damage, and increased cisplatin sensitivity. Endoglin downregulation led to a decrease in expression of several DNA repair genes, including NBN, NTHL1, BARD1, H2AFX and SIRT1, and an increase in expression of DDIT3 and PPP1R15A. BARD1-specific downregulation, in turn, led to a significant decrease in BRCA1 expression, likely through ubiqutinylation. In vivo, anti-endoglin treatment decreased tumor weight in both ES2 and HeyA8MDR models when compared to control (35-41% reduction, p<0.05). Combination treatment with anti-Endoglin siRNA and carboplatin was associated with even greater inhibitory effect compared to control (58-62% reduction, p<0.001). Conclusions: Targeting endoglin improves platinum sensitivity of ovarian cancer cells both in vivo and in vitro. Endoglin downregulation induces DNA damage at multiple levels, one of which is through decreased expression of BARD1 and BRCA1. Anti-endoglin therapies should be pursued further as their development would allow dual treatment of both tumor angiogenesis and an aggressive subset of chemoresistant tumor cells. Citation Format: Angela J. Ziebarth, Somaria Nowsheen, Adam D. Steg, Monjri M. Shah, Ashwini A. Katre, Zachary C. Dobbin, Anil K. Sood, Michael G. Conner, Eddy S. Yang, Charles N. Landen. Endoglin (CD105) is a target for ovarian cancer cell-specific therapy through induction of DNA damage. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5475. doi:10.1158/1538-7445.AM2013-5475 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 1728: Targeting hedgehog reverses taxane resistance by Gli-dependent and independent mechanisms in ovarian cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Objective: Recent studies have implicated hedgehog signaling in the formation and continued growth of a variety of malignancies, including ovarian cancer. Several inhibitors of the hedgehog pathway have been identified that block the activity of the Smoothened (Smo) receptor. The goal of this study was to determine the in vitro and in vivo effects of Smo antagonists alone and in combination with chemotherapy in ovarian cancer. Methods: Expression of hedgehog signaling components (Smo, Gli1 and Gli2) was assessed in 3 pairs of parental and chemotherapy-resistant ovarian cancer cell lines (A2780ip2/A2780cp20, SKOV3ip1/SKOV3TRip2, HeyA8/HeyA8MDR) using Western blot and qPCR. Cell lines were exposed to increasing concentrations of two different Smo antagonists (Cyclopamine, LDE225) alone and in combination with carboplatin, paclitaxel, adriamycin, and topotecan. Selective knockdown of Smo, Gli1 and Gli2 was achieved using siRNA constructs. Cell viability was assessed by MTT assay and PARP cleavage was used as an indicator of apoptosis. SKOV3TRip2 orthotopic xenografts were treated with vehicle, LDE225, paclitaxel or combination therapy for 5 weeks. Tumor weight for each treatment group was measured and compared using students t-test. Results: Expression of Smo and Gli1 was high in A2780ip2/A2780cp20, moderate in SKOV3ip2/SKOV3TRip2 and low/absent in HeyA8/HeyA8MDR. Gli2 was high in SKOV3ip2/SKOV3TRip2, moderate in A2780ip2/A2780cp20 and low in HeyA8/HeyA8MDR. Response to cyclopamine and LDE225 varied among the cell lines examined with IC50s ranging from 7.5 to >20 µM. Both agents sensitized chemotherapy-resistant cell lines to paclitaxel (5- to 26-fold, including Smo[low]/Gli1[neg] HeyA8MDR), but not to carboplatin, adriamycin, or topotecan. Selective knockdown of Gli1 and Gli2 resulted in taxane sensitization only in Gli1/2-high A2780cp20 cells (2- to 8-fold). A decrease in acetyl-α-tubulin confirmed microtubule-specific effects of Smo targeting, supporting the taxane specificity of this effect. In vivo, SKOV3TRip2 xenografts treated with LDE225 or paclitaxel alone had slightly less tumor burden than the control group (reduced by 28.1%, p=0.42 and 32.0%, p=0.40, respectively). Those treated with combined LDE225 and paclitaxel, however, had significantly less tumor burden than those treated with vehicle (70.5% reduction, p=0.015). Conclusions: Targeting the hedgehog pathway decreases cell viability and increases taxane sensitivity in taxane-resistant ovarian cancer models. Interestingly, these effects were noted even in cells with little constitutive hedgehog activity. This suggests both Gli-dependent and -independent mechanisms contribute to taxane resistance, expanding the potential use of hedgehog inhibitors to all taxane-resistant tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1728. doi:10.1158/1538-7445.AM2011-1728

Abstract 1649: The E3 ubiquitin ligase EDD regulates platinum resistance and is a novel therapeutic target for epithelial ovarian cancer

Introduction: Ovarian cancer is treated with a combination of surgery and chemotherapeutics, including taxol and platinum agents. A major difficulty in eradicating these tumors is the selection of drug resistant cells. EDD (E3-ubiquitin ligase identified by Differential Display) is a 300 kDa E3 ubiquitin ligase that is overexpressed in 84% of recurrent, platinum-resistant ovarian cancers, but is rare in benign in borderline tumors. This correlation with chemoresistance suggests that EDD may be involved in mediating platinum resistance and/or tumor survival. Experimental Procedures: EDD protein knockdown was accomplished using siRNA (small interfering RNA) and shRNA (short hairpin RNA) for transient or stable knockdown, respectively, in ovarian cancer cell lines. Apoptosis and sensitivity to cisplatin were measured using MTS assay, propidium iodide staining, and western blotting for PARP and caspase 3 cleavage. Cisplatin resistance by EDD overexpression was determined by transient transfection of GFP, FLAG-EDD or the ubiquitin ligase deficient FLAG-EDD-C2768A into COS-7 cells followed by cisplatin treatment and apoptosis measurement in transfected cells by the TACS 2 TdT-Blue Label Kit followed by microscopy. Mice with intraperitoneal xenografts of ES2 and A2780ip2 cells were treated once weekly with cisplatin and twice weekly with control or EDD siRNA. Results: EDD was overexpressed in 5 of 7 ovarian cancer cell lines compared to benign IOSE cells. EDD overexpression in COS-7 cells was sufficient to protect from cisplatin-induced apoptosis and the induced resistance was dependent upon EDD ubiquitin ligase activity (relative apoptosis: GFP = 1; EDD = 0.42, EDD-C2768A = 1.23). Stable knockdown of EDD increased cisplatin sensitivity in ES2 cells (IC50 = 37 μM for control, 9 μM for EDD shRNA). EDD siRNA alone induced cleavage of PARP and caspase 3 compared to control siRNA and enhanced cleavage of both in the presence of cisplatin. Mouse xenograft studies demonstrated that nanoliposomal delivery (DOPC) of EDD siRNA showed a trend towards reduced tumor compared to control siRNA. In vivo, mice treated with EDD siRNA by nanoliposomal delivery (DOPC) had a trend towards less tumor than those treated with control siRNA (27.7% reduction in ES2, 42.5% in A2780ip2, p = 0.23 and 0.19 respectively). Mice treated with combined EDD siRNA and cisplatin had significantly less tumor than controls (77.9% reduction in ES2, 75.9% in A2780ip2, p = 0.004 and 0.042, respectively) and cisplatin alone (60.3-64.4% reduction, p = 0.035). Conclusions: EDD overexpression is sufficient to promote cisplatin resistance, dependent upon its E3 ubiquitin ligase activity. Targeting EDD expression induces apoptosis and cisplatin. sensitivity in vitro and in vivo. Targeting EDD may be an effective treatment for platinum-resistant ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1649. doi:10.1158/1538-7445.AM2011-1649