Abstract
BackgroundOvarian cancer is the leading cause of gynecologic cancer death in the United States despite effective first-line systemic chemotherapy. Cancer stem cells (CSCs) retain the ability to self-renew and proliferate and may be a means of harboring disease that evades standard treatment strategies. We previously performed a high-throughput screen to assess differential protein expression in ovarian CSCs compared to non-CSCs and observed that Thy-1 was more highly expressed in CSCs. Our primary aim was to validate Thy-1 (CD90) as a cancer stem cell (CSC) marker in epithelial ovarian cancer (EOC), correlate with clinical outcomes, and assess as a potential therapeutic target.ResultsKaplan Meier (KM) Plotter data were correlated with survival outcomes. Quantitative real-time PCR, flow cytometry, and immunoblots assessed RNA and protein expression. Limiting dilution assays assessed self-renewal capacity and proliferation assays assessed proliferative capacity. RNA in-situ hybridization was performed on patient specimens to assess feasibility. Thy-1 (CD90) is more highly expressed in ovarian CSCs than non-CSCs, in EOC compared to benign ovarian epithelium (P < 0.001), and is highest in serous EOC (P < 0.05). Serous ovarian cancers with high Thy-1 expression have poorer outcomes (median PFS 15.8 vs. 18.3 months, P = 0 < 0.001; median OS 40.1 v. 45.8 months, P = 0.036). Endometrioid ovarian cancers with high Thy-1 have poorer PFS, but no difference in OS (upper quartile PFS 34 v. 11 months, P = 0.013; quartile OS not reached, P = 0.69). In vitro, Thy-1 expression is higher in CSCs versus non-CSCs. EOC cells with high Thy-1 expression demonstrate increased proliferation and self-renewal. Thy-1 knockdown in EOC cells decreases proliferative capacity and self-renewal capacity, and knockdown is associated with decreased expression of stem cell transcription factors NANOG and SOX2. RNA in situ hybridization is feasible in ovarian cancer tissue specimens.ConclusionsThy-1 is a marker of ovarian CSCs. Increased expression of Thy-1 in EOC predicts poor prognosis and is associated with increased proliferative and self-renewal capacity. Thy-1 knockdown decreases proliferative and self-renewal capacity, and represents a potential therapeutic target.
Highlights
Ovarian cancer is the leading cause of gynecologic cancer death in the United States despite effective first-line systemic chemotherapy
Thy-1 is more highly expressed in ovarian cancer stem cell (CSC) compared to non-CSCs We previously described and validated a NANOG promotor-driven Green Fluorescent Protein (GFP) reporter system for isolating ovarian CSCs (GFP-positive) from non-CSCs (GFP-negative) and subsequently reported our finding that CD55, a cell surface complement inhibitor, was differentially expressed in ovarian CSCs as compared to non-CSCs [9, 12]
From a total of 242 cell surface proteins that were included in this high-throughput screen, we identified Thy-1 was more highly expressed in ovarian CSCs as compared to non-CSCs in A2780 cisplatin-naïve ovarian cancer cells (Fig. 1a, b)
Summary
Ovarian cancer is the leading cause of gynecologic cancer death in the United States despite effective first-line systemic chemotherapy. We previously performed a high-throughput screen to assess differential protein expression in ovarian CSCs compared to non-CSCs and observed that Thy-1 was more highly expressed in CSCs. Our primary aim was to validate Thy-1 (CD90) as a cancer stem cell (CSC) marker in epithelial ovarian cancer (EOC), correlate with clinical outcomes, and assess as a potential therapeutic target. We previously transduced ovarian cancer cells (A2780) with a NANOG-GFP reporter system to identify ovarian CSCs based on GFP intensity [8, 9] Using this platform, we performed a high-throughput flow cytometry screen to compare expression of 242 cell surface markers in ovarian CSCs (GFP-positive) and nonCSCs (GFP-negative) and identified CD55 as a CSC marker and a driver of self-renewal and chemoresistance pathways [10,11,12]. Our high-throughput screen identified a second protein that was more highly expressed in CSCs, Thy-1
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