Published OnlineFirst July 27, 2011; DOI: 10.1158/0008-5472.CAN-10-2719 Cancer Therapeutics, Targets, and Chemical Biology Research Biological Roles of the Delta Family Notch Ligand Dll4 in Tumor and Endothelial Cells in Ovarian Cancer Wei Hu1, Chunhua Lu1, Hee Dong Han1, Jie Huang1, De-yu Shen1, Rebecca L. Stone1, Alpa M. Nick1, Mian M.K. Shahzad1, Edna Mora1, Nicholas B. Jennings1, Sun Joo Lee1, Ju-Won Roh1, Koji Matsuo1, Masato Nishimura1, Blake W. Goodman1, Robert B. Jaffe6, Robert R. Langley2, Michael T. Deavers3, Gabriel Lopez-Berestein4, Robert L. Coleman1, and Anil K. Sood1,3,5 Abstract 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 compart- ments of ovarian cancer and that targeting DLL4 in combination with anti-VEGF treatment might improve outcomes of ovarian cancer treatment. Cancer Res; 71(18); 1–12. Ó2011 AACR. Introduction ing the tumor vasculature is a particularly attractive strategy because of the presumed genetic stability of endothelial cells Ovarian cancer remains the leading cause of death from a (4). The recent success of antiangiogenic therapy with bev- gynecologic malignancy among women in the United States. acizumab in solid tumors, including ovarian cancer, has Although tumor-reductive surgery and taxane- and platinum- confirmed the clinical viability of this approach (5). Despite based chemotherapy regimens are effective treatments for initial responses, however, most patients eventually experi- primary disease in the majority of patients with this cancer, ence tumor progression resulting in their death, mainly due to recurrence is common and often leads to death. New thera- development of drug resistance. The precise molecular peutic agents are needed to improve survival rates and, mechanisms underlying clinical resistance to anti-VEGF eventually, to cure this deadly disease. therapies are not well understood. Drug resistance can arise The progressive growth of primary tumor and metastasis is because of alterations in pharmacokinetics, cancer dependent on angiogenesis. VEGF, also known as vascular cellÀspecific abnormalities, and/or alterations in the tumor permeability factor, plays a pivotal role in developmental, microenvironment (6–8). Therefore, additional targets for physiologic, and pathologic neovascularization (1–3). Target- antiangiogenesis strategies are urgently needed. The Notch signaling pathway has recently been implicated in tumor angiogenesis, including vessel maturation, pericyte 1 2 Authors' Affiliations: Departments of Gynecologic Oncology, Cancer recruitment, branching and cell differentiation, proliferation, Biology, 3Pathology and 4Experimental Therapeutics, 5Center for RNAi and non-coding RNA, The University of Texas MD Anderson Cancer survival, and apoptosis. In mammalian cells, this pathway Center, Houston, Texas; and 6Center for Reproductive Sciences, Univer- comprises 5 transmembrane Notch ligands (Jagged 1, Jagged 2, sity of California, San Francisco, California and Delta-like ligands [Dll] 1, 3, and 4) and 4 Notch receptors Note: Supplementary data for this article are available at Cancer Research (Notch 1–4). Ligand receptor binding leads to cleavage via Online (http://cancerres.aacrjournals.org/). intramembrane proteolysis by g-secretase and subsequent W. Hu and C. Lu contributed equally to this work. translocation from the cell membrane to the nucleus. The Corresponding Author: Anil K. Sood, Departments of Gynecologic Notch intracellular domain interacts with transcription fac- Oncology and Cancer Biology, The University of Texas MD Anderson Cancer Center, 1155 Herman Pressler Blvd., Unit 1352, tors to regulate transcription of the basic helix-loop-helix Houston, TX 77030. Phone: 713-745-5266; Fax: 713-792-7586; E-mail: proteins hairy/enhancer of split (HES) and HES-related pro- [email protected] teins (HEY). doi: 10.1158/0008-5472.CAN-10-2719 Dll4 is an endothelium-specific ligand expressed at sites of Ó2011 American Association for Cancer Research. vascular development and angiogenesis. Dll4 expression has www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2011 American Association for Cancer Research. Published OnlineFirst July 27, 2011; DOI: 10.1158/0008-5472.CAN-10-2719 Hu et al. previously been shown to be upregulated within the vascula- ical vein endothelial cells (HUVEC) were purchased from ture of breast, renal, and bladder cancers (9). It has been Cambrex and maintained with heparin and gentamicin/am- suggested that VEGF induces Dll4/Notch signaling whereas photericin B. Dll4/Notch signaling modulates the VEGF pathway, that Dll4 and VEGF merge to be the "yin and yang" of angiogenesis (10– Dll4 gene silencing in MOEC and A2780 ovarian cancer 12). Recently, some studies have reported that Dll4 blockade cells inhibits tumor growth by inducing nonproductive angiogen- Nonsilencing control siRNA and human and mouse Dll4 esis manifested by increased vascular density and decreased siRNA sequences were obtained from Sigma-Aldrich. The perfusion in tumors. The precise mechanism of inhibition has nonsilencing siRNA did not share sequence homology with not been elucidated, however, and the biological significance any known human mRNA (based on a BLAST search). The of Dll4 in ovarian cancer, especially VEGF-resistant ovarian sequences are included in supplementary Table S3. For in vitro cancer, is not well understood. transfection studies, N-TER Transfection Kit (Sigma-Aldrich) In this study, we examined the clinical, functional, and was used per the manufacturer's guidelines. biological significance of Dll4 in ovarian cancer angiogenesis and tumor progression by using therapeutically relevant Dll4 Reverse transcriptase PCR silencing. Our findings indicate that Dll4 is a potential target Relative expression of Jag 1, Jag2, Dll1, Dll3, Dll4, Notch1, for antiangiogenic therapeutic strategies. Notch2, Notch3, and Notch4 in cells representing ovarian cancer (HeyA8, SKOV3ip1, OVAR3, A2774, IGROV), endothe- Materials and Methods lium (HUVEC, MOEC), and pericyte-like (10T1/2) and non- transformed ovarian surface epithelial cells (HIO180) was Human ovarian cancer specimens determined by reverse transcriptase PCR (RT-PCR). Each Following approval by the Institutional Review Board, 84 RT-PCR reaction used 5 mg total RNA isolated from treated paraffin-embedded epithelial ovarian cancer specimens with cells using the RNeasy Mini Kit (Qiagen). Primer sequences, available clinical outcome data and confirmed diagnosis by a size of PCR products, and annealing temperature are given in board-certified gynecologic pathologist were obtained from supplementary Tables S1and S2. Real-time quantitative RT- the Gynecologic Oncology tumor bank of The University of PCR was done in an ABI 7500 Sequence Detection System Texas MD Anderson Cancer Center. All 84 cases were diag- (Applied Biosystems). The SensiMix SYBR Low-ROX Kit was nosed between 1986 and 2003 following primary cytoreductive used (Bioline USA Inc.).The relative quantification (RQ) was ÀDDC surgery. Slides of tumor samples were obtained for Dll4 calculated by 2 T. expression analysis. Clinical variables obtained for correlative analyses included age at diagnosis, tumor stage and grade, and Western blot analysis vital status of patients relative to disease-specific survival at Western blot analysis was done as previously reported the time of chart review. An additional 24 paraffin-embedded (16, 17). epithelial ovarian cancer specimens from patients who were treated with an anti-VEGF agent (aflibercept or bevacizumab) DNA extraction and methylation analysis were obtained from the Gynecologic Oncology tumor bank. DNA was extracted from MOEC using standard phenol– chloroform methods. MOEC were treated with immobilized Immunohistochemical staining Dll4 (1, 10, or 20 mg/mL). Other MOEC were treated with the Immunohistochemical staining for Dll4 (1:200 dilution) was demethylating agent azacytidine (AZA; 25 mmol/L), immobi- quantified by 2 investigators in a blinded fashion on the basis lized Dll4 (10 mg/mL), or a combination of the two for 48 of percentage of positively stained tumor cells and staining hours. Methylation status was determined by methylation- intensity. The Dll4 antibody, which reacts with both mouse specific PCR using a methylation kit (EZ-96 gold; Zymo and human Dll4, was obtained from Rockland Immunochem- Research). MethPrimer software was used for prediction of icals for Research. For