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SummaryWhile recombinant human erythropoietin (rhEpo) has been widely used to treat anemia in cancer patients, concerns about its adverse effects on patient survival have emerged. A lack of correlation between expression of the canonical EpoR and rhEpo’s effects on cancer cells prompted us to consider the existence of an alternative Epo receptor. Here, we identified EphB4 as an Epo receptor that triggers downstream signaling via STAT3 and promotes rhEpo-induced tumor growth and progression. In human ovarian and breast cancer samples, expression of EphB4 rather than the canonical EpoR correlated with decreased disease-specific survival in rhEpo-treated patients. These results identify EphB4 as a critical mediator of erythropoietin-induced tumor progression and further provide clinically significant dimension to the biology of erythropoietin.

Metastasis is a complex, multistep process that begins with the epithelial–mesenchymal transition (EMT). Circulating tumor cells (CTC) are believed to have undergone EMT and thus lack or express low levels of epithelial markers commonly used for enrichment and/or detection of such cells. However, most current CTC detection methods target only EpCAM and/or cytokeratin (CK) to enrich epithelial CTCs, resulting in failure to recognize other, perhaps more important, CTC phenotypes that lack expression of these markers. Here, we describe a population of complex aneuploid CTCs that do not express CK or CD45 antigen in patients with breast, ovarian, or colorectal cancer. These cells were not observed in healthy subjects. We show that the primary epithelial tumors were characterized by similar complex aneuploidy, indicating conversion to an EMT phenotype in the captured cells. Collectively, our study provides a new method for highly efficient capture of previously unrecognized populations of CTCs. Significance: Current assays for CTC capture likely miss populations of cells that have undergone EMT. Capture and study of CTCs that have undergone EMT would allow a better understanding of the mechanisms driving metastasis. Cancer Discovery; 1(7); 580–86. ©2011 AACR. This article is highlighted in the In This Issue feature, p. 539

Emerging evidence suggests that the Notch/Delta-like ligand 4 (Dll4) pathway may offer important new targets for antiangiogenesis approaches. In this study, we investigated the clinical and biological significance of Dll4 in ovarian cancer. Dll4 was overexpressed in 72% of tumors examined in which it was an independent predictor of poor survival. Patients with tumors responding to anti-VEGF therapy had lower levels of Dll4 than patients with stable or progressive disease. Under hypoxic conditions, VEGF increased Dll4 expression in the tumor vasculature. Immobilized Dll4 also downregulated VEGFR2 expression in endothelial cells directly through methylation of the VEGFR2 promoter. RNAi-mediated silencing of Dll4 in ovarian tumor cells and tumor-associated endothelial cells inhibited cell growth and angiogenesis, accompanied by induction of hypoxia in the tumor microenvironment. Combining Dll4-targeted siRNA with bevacizumab resulted in greater inhibition of tumor growth, compared with control or treatment with bevacizumab alone. Together, our findings establish that Dll4 plays a functionally important role in both the tumor and endothelial compartments of ovarian cancer and that targeting Dll4 in combination with anti-VEGF treatment might improve outcomes of ovarian cancer treatment. Cancer Res; 71(18); 6030–9. ©2011 AACR.

Purpose: Jagged1, a Notch ligand, is expressed on both tumor epithelial and endothelial cells and therefore may be amenable to dual targeting of the tumor stroma and malignant cell compartments of the tumor microenvironment. Experimental Design: We describe in vitro effects of targeting of Jagged1 on ovarian cancer cells and in vivo effects of independent targeting of stromal and malignant cell Jagged1 using species-specific human or murine siRNA constructs incorporated into chitosan nanoparticles and delivered intravenously in an orthotopic mouse model. Results: Jagged1 expression was prominent in SKOV3ip1 and IGROV-AF1, and significantly overexpressed in SKOV3TRip2, a taxane-resistant SKOV3 subclone. Jagged1 silencing with siRNA decreased cell viability and reversed taxane chemoresistance. In two different orthotopic ovarian cancer models, treatment with anti-human Jagged1 siRNA-CH reduced growth by 54.4% to 58.3% and with anti-murine Jagged1 siRNA-CH reduced growth by 41.7% to 48.8%. The combination of both species-specific constructs reduced tumor weight by 87.5% to 93.1% and sensitized SKOV3TRip2 tumors to docetaxel in vivo. Tumors showed reduced microvessel density with anti-murine Jagged1 constructs and decreased proliferation with anti-human Jagged1 siRNAs-CH. In addition, we show that Jagged1 downregulation does not sensitize cells to taxanes through a reduction in MDR1 expression, but at least in part by cross-talk with the GLI2 mediator of the Hedgehog pathway. Conclusions: Jagged1 plays dual roles in cancer progression through an angiogenic function in tumor endothelial cells and through proliferation and chemoresistance in tumor cells. Dual inhibition represents an attractive therapeutic strategy for ovarian and potentially other malignancies. Clin Cancer Res; 17(17); 5674–85. ©2011 AACR.

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.

OBJECTIVE. Aldehyde dehydrogenase-1 (ALDH1) expression characterizes a subpopulation of cells with enhanced tumor initiating and differentiating properties in some cancers. We have examined the association of ALDH1 with chemoresistance and whether downregulation of ALDH1 sensitizes cells to chemotherapy in models of ovarian cancer. METHODS. Microarray profiling was performed on SKOV3ip1 and the taxane-resistant SKOV3TRip2 cell lines. Primary ovarian cancer xenografts with and without cisplatin exposure were examined for selection of ALDH1-positive cells. Small interfering RNA (siRNA) was used to downregulate ALDH1 in vitro, and in vivo by incorporation into neutral DOPC liposomes, for evaluation of chemosensitization in an orthotopic model of ovarian cancer. RESULTS. Microarray analysis found 29 genes upregulated and 18 genes downregulated by more than 10-fold when comparing the taxane-resistant SKOV3TRip2 ovarian cancer line compared to its parental SKOV3ip1 line. Included among these was a 92.7-fold higher ALDH1 signature. Increased expression and activity of ALDH1 was confirmed by Western blot and the ALDEFLUOR assay (58% of cells ALDH1-active). In primary ovarian cancer xenografts in NOD-Scid mice, cisplatin treatment resulted in an increase in ALDH1-positive cells, from a baseline of 1% to 38% with therapy (p CONCLUSIONS. ALDH1 expression is associated with taxane and cisplatin chemoresistance in ovarian cancer cell lines. ALDH1 expression can be induced by cisplatin treatment in vivo, and targeting ALDH1 sensitizes resistant cell lines to chemotherapy. This enzyme may be important for identification and targeting the chemoresistant population in ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4288.

SummaryAlthough 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.