A79
Over-expression of Polycomb Repressive Complex 2 (PRC2) genes in rare chemoresistant side populations of ovarian cancer
Sian Rizzo1, Alessandra Silva1, Jennifer M. Quinn1, Wei Dai2, Constanze Zeller2, Stanley B. Kaye1, David L. Hudson1, Robert Brown1
1Institute of Cancer Research, Sutton, UK, 2Ovarian Cancer Action Centre, Imperial College, London, UK
Background
Subpopulations of tumour cells include so-called Side Populations (SPs), which are capable of drug-efflux and over-express ABC drug-transporters have been identified in a variety of cancers and are proposed to have a key role in the sustained growth of tumours and acquired drug resistance. We aim to characterise ovarian drug resistant tumour-sustaining cells with the objective of identifying novel therapeutic targets.
Method
SPs and non-SPs were isolated using differential Hoechst33342 dye uptake and fluorescence-activated cell sorting (FACS). Gene expression profiles were generated using Affymetrix Human Genome U133plus2.0 chip arrays. Target genes were knocked down using siRNAs and phenotype assessed using qRT-PCR and FACS.
Results
We found 0-8% SPs in ovarian cancer cell lines (n=9) and 0-5% in patient ascites (n=10), with a trend for increased SP in patient samples post-chemotherapy. Carboplatin treatment of IGROV1 ovarian cancer cells lead to a 3-fold increase in percentage of SP cells, while untreated SPs showed 3-fold and 2-fold increased resistance to carboplatin and taxol, respectively, compared to non-SPs. IGROV1 SPs showed significant increase in spheroid-formation compared to non-SP and increased tumour take when injecting 50 or 100 cells into NOD/SCID mice. A NOS signature (Notch/Oct4/Sox2 regulated genes), associated with embryonic stem cells, was found to be significantly over-expressed in the SP compared to non-SP expression profile. Polycomb Repressive Complex 2 (PRC2) genes were also significantly over-represented in SP, while PRC2-repressed target genes were significantly under-represented. Knock-down of key PRC2 components EZH2 plus EZH1 lead to loss of SP in multiple ovarian cell lines.
Conclusion
Rare drug resistant subpopulations with tumour sustaining capability can be identified in ovarian cancer cells. PRC2 members are over-expressed in SP cells from cell lines and tumours and siRNA knock-down suggests EZH2 and EZH1 as potential targets for development of drugs that target the SP population.
