Identification of a Potential Ovarian Cancer Stem Cell Gene Expression Profile from Advanced Stage Papillary Serous Ovarian Cancer

Vinod Vathipadiekal,Peter V Hauschka,Samuel C Mok,Deepa Saxena,Laurent Ozbun,Michael J Birrer,Xin-Yuan Guan

doi:10.1371/journal.pone.0029079

Abstract

Identification of gene expression profiles of cancer stem cells may have significant implications in the understanding of tumor biology and for the design of novel treatments targeted toward these cells. Here we report a potential ovarian cancer stem cell gene expression profile from isolated side population of fresh ascites obtained from women with high-grade advanced stage papillary serous ovarian adenocarcinoma. Affymetrix U133 Plus 2.0 microarrays were used to interrogate the differentially expressed genes between side population (SP) and main population (MP), and the results were analyzed by paired T-test using BRB-ArrayTools. We identified 138 up-regulated and 302 down-regulated genes that were differentially expressed between all 10 SP/MP pairs. Microarray data was validated using qRT-PCR and17/19 (89.5%) genes showed robust correlations between microarray and qRT-PCR expression data. The Pathway Studio analysis identified several genes involved in cell survival, differentiation, proliferation, and apoptosis which are unique to SP cells and a mechanism for the activation of Notch signaling is identified. To validate these findings, we have identified and isolated SP cells enriched for cancer stem cells from human ovarian cancer cell lines. The SP populations were having a higher colony forming efficiency in comparison to its MP counterpart and also capable of sustained expansion and differentiation in to SP and MP phenotypes. 50,000 SP cells produced tumor in nude mice whereas the same number of MP cells failed to give any tumor at 8 weeks after injection. The SP cells demonstrated a dose dependent sensitivity to specific γ-secretase inhibitors implicating the role of Notch signaling pathway in SP cell survival. Further the generated SP gene list was found to be enriched in recurrent ovarian cancer tumors.

Highlights

Epithelial ovarian cancer is the fifth leading cause of death in women in the United States
The positive CA 125 staining using FACS confirmed the ovarian origin of cells isolated from ascites (Figure S1) and these cells were analyzed for side population (SP) and main population (MP) cells using Hoechst and/or Rhodamine 123 (Rho) staining
This study reports a SP cell gene expression signature from isolated SP of fresh ascites obtained from women with high-grade advanced stage papillary serous ovarian adenocarcinoma using Affymetrix U133 Plus 2.0 microarray platform

Summary

Introduction

Epithelial ovarian cancer is the fifth leading cause of death in women in the United States. There is increasing evidence that small populations of cells within tumors called cancer stem cells (CSC) contributes to tumor maintenance and progression and are intrinsically resistant to therapies designed to destroy rapidly dividing cells [3,4,5,6,7]. Experiments performed on human acute myeloid leukemia [15] and solid tumors [8,9] show that CSCs display three functional characteristics: 1) they have the tumorigenic potential to form tumors when injected into nude mice, 2) they express distinct surface markers allowing for reproducible and differential purification, and 3) they have the ability to recreate the full phenotypic heterogeneity of the parent tumor [16,17]. The definition for CSC is a functional one and shares two important functional characteristics with normal stem cells: selfrenewal and differentiation [3,7]

Methods

Results

Conclusion