Purpose Within heterogeneous tumors, subpopulations often labeled cancer stem cells (CSCs) 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, while 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 to 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 demonstrating 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.

The hedgehog (HH) pathway has been implicated in the formation and maintenance of a variety of malignancies, including ovarian cancer; however, it is unknown whether HH signaling is involved in ovarian cancer chemoresistance. The goal of this study was to determine the effects of antagonizing the HH receptor, Smoothened (Smo), on chemotherapy response in ovarian cancer. Expression of HH pathway members was assessed in 3 pairs of parental and chemotherapy-resistant ovarian cancer cell lines (A2780ip2/A2780cp20, SKOV3ip1/SKOV3TRip2, HeyA8/HeyA8MDR) using qPCR and Western blot. 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 demonstrated higher expression (>2-fold, p<0.05) of HH signaling components compared to 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 appeared 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-HH and chemotherapies could provide a useful therapeutic strategy for ovarian cancer.

Purpose Endoglin (ENG, 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 qPCR. 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, anti-endoglin treatment decreased tumor weight in both ES2 and HeyA8MDR models when compared to control (35-41% reduction, p<0.05). Endoglin inhibition with carboplatin was associated with even greater inhibitory effect when compared to 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. Anti-endoglin 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.

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.

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.