2012;366:1382C1392. and were enriched in xenografts residual after platinum therapy. Low dose SGI-110 reduced the stem-like properties of ALDH+ cells, including their tumor initiating capacity, resensitized these OCSCs to platinum, and induced re-expression of differentiation-associated genes. Maintenance treatment with SGI-110 after carboplatin inhibited OCSC growth, causing global tumor hypomethylation and decreased tumor progression. Our work offers preclinical evidence that epigenome-targeting strategies have the potential to delay tumor progression by re-programming residual cancer stem-like cells. Further, the results suggest that SGI-110 might be administered in combination with platinum to prevent the development of recurrent and chemoresistant ovarian cancer. INTRODUCTION Epithelial ovarian cancer (OC) causes Thiostrepton more deaths than any other female reproductive tract cancer (1,2). The majority of women diagnosed with advanced-stage epithelial OC experience tumor recurrence associated with the development of chemoresistance, and platinum-resistant OC is uniformly fatal (3). A new paradigm explaining tumor relapse involves the persistence of cancer stem cells which were characterized in several solid tumors, including OC (4C6). Thiostrepton While chemotherapy may succeed initially at decreasing the size and number of tumors, it leaves behind residual malignant cells, which we hypothesize are enriched in tumor progenitors or cancer stem cells. Ovarian cancer stem cells (OCSCs) have been isolated from established OC cell lines, ascites, primary and metastatic tumors (4,7,8). They share several characteristics with normal stem cells, including the ability to form anchorage-independent spherical aggregates, express stem cell markers, undergo membrane efflux, form clones in culture and in addition exhibit enhanced tumor-forming ability (9). Although a number of technical approaches have been successfully used to isolate OCSCs (sphere-forming, cell surface Thiostrepton markers, stem cell gene reporter assays), the use of an assay measuring aldehyde dehydrogenase isoform 1 (ALDH) enzymatic activity has been recently proposed and is used to define CSCs in multiple other tumor types (10,11). Ovarian CSCs are hypothesized to be largely (or entirely) responsible for emergence of chemoresistant tumors, because they possess many of the phenotypes associated with drug resistance (e.g., enhanced DNA repair, diminished apoptotic responses, increased efflux mechanisms, quiescent state) (4,12). Moreover similarly to normal embryonic or tissue stem cells, CSC are believed to harbor a significantly altered epigenome (6,13), and it has been hypothesized that DNA hypomethylating agents could reset these cells toward differentiation (14). Indeed, several hypomethylating providers were originally characterized as inducers of malignancy cell differentiation (6,15). However, it has become obvious that hypomethylating providers or additional epigenetic modulators only cannot eradicate relapsed tumors. Pre-clinical studies from our and additional groups have established the rationale for combining DNA methylation inhibitors with existing chemotherapeutic providers to overcome acquired drug resistance in OC (16C20). Based on those studies, we recently completed a phase II trial using a DNA methylation inhibitor like a re-sensitizer to traditional chemotherapy in individuals with recurrent OC and showed that this combination has medical and biological activity (21), justifying additional rationally designed epigenetic treatment strategies in OC. Based on the above considerations, we hypothesized that hypomethylating providers, in combination with chemotherapeutics, may target drug-resistant OCSCs, probably leading to tumor eradication. In the current study, we isolated and characterized ALDH(+) OCSC from OC cell lines and human being tumors. ALDH(+) cells were significantly more chemoresistant and tumorigenic compared to ALDH(?) cells in orthotopic tumor initiating assays. Treatment with SGI-110, a second-generation DNA methyltransferase inhibitor (DNMTI), resensitized OCSCs to platinum. A model recapitulating the emergence of recurrent tumors showed an increased percentage of ALDH(+) OCSCs in residual tumors after platinum. Maintenance therapy with SGI-110 during platinum-induced remission inhibited the emergence of platinum resistant tumors. We suggest that epigenomic focusing on using SGI-110 may be useful like a maintenance medical strategy after platinum-based therapy in OC. MATERIALS AND METHODS Cell lines, patient samples, tradition conditions and reagents OC cell lines (A2780, A2780_CR5, SKOV3) MYH10 were managed in RPMI 1640 medium (Invitrogen, Carlsbad, CA) with health supplements as explained previously (22) and see supplemental methods. Cisplatin-resistant variant A2780_CR5 was founded from five-round IC70 survival monoclonal-selection by continuous exposure to increasing concentration of cisplatin (22). A2780 and SKOV3 OC cells were authenticated in 2012 by ATCC (Manassas, VA). Advanced Thiostrepton high grade serous ovarian tumors were surgically collected (IRB approved protocol IUCRO-0280), enzymatically disassociated and cultured, as previously described.
