Abstract 4261: Epigenetic reprogramming affects malignant properties of human liver cancer cells

Background: A plastic phenotype is a significant hallmark of aggressive tumors and embryonic stem cells (ESC). Cancer plasticity is dynamically regulated both by cell microenvironment and epigenetic machinery therefore playing important roles in transcriptional reprogramming and cell fate stable changes. Objective: We have explored the relationship between epigenetic alterations and local microenvironment in regulation of cancer stem cells (CSCs) using Zebularine (Zeb), a DNA-methyltransferase1 inhibitor, in combination with the modulation of cellular density in culture. Methods: Seven human hepatoma cell lines, including two primary HCC cell lines, were plated at high (HD) and low density (LD) and grown in the presence or absence of Zeb for 3 days. After, cells were propagated in serum-free condition for evaluation of sphere-forming capacity. Primary tumor-spheres were passaged every 10 days to produce generation 1 (G1) trough G5. Spheres at different generations were examined for differences in gene expression and tumorigenic capacity in vivo. The sphere-derived mRNA and microRNA gene expression signatures generated for different passages were integrated with human HCC database and tested for their prognostic value by Kaplan-Meier. Results: Cells grown under LD failed to form spheres when placed in non-adherent growth conditions. Cells cultured at HD displayed a sphere forming potential at G1 but were unable to maintain extended sphere forming capacity regardless of Zeb pre-treatment. In contrast, Zeb-treated LD (LDZ) cells showed unique features in terms of self renewal potential both in vitro and vivo. LDZ-derived spheres were more proliferative as indicated by Ki67 staining whereas HDZ-spheres showed activation of apoptotic pathway and dowregulation of NF-kB protein levels. LDZ also maintained a higher expression of CSC related markers (CD133, CD44, EpCAM) as well as key factors involved in ESC self-renewal and pluripotency (Bmi1 and αSuz12, subunit of distinct polycomb complexes, PRC1 and PRC2). Also, LDZ-derived spheres consistently expressed higher levels of DNMT1 and DNMT3a. In addition, LDZ spheres exhibited epithelial-mesenchymal transition attributes as judged by the upregulation of β-catenin, ZEB1, VIM and SNAL1 markers as compared to HD spheres. Finally, only G1 LDZ-spheres derived cells showed higher in vivo tumorigenicity and could give rise to brain metastasis. Consistently, global transcriptome analysis confirmed that LDZ sphere signature was enriched in oncogenic molecules associated with metastasis, invasion and resistance to apoptosis, including SMAD2, CD133, CD44 and NF-kB and was able to predict the clinical outcome of liver cancer patients. Conclusions: Epigenetic reprogramming of liver cancer cell lines induced by a combined modulation of DNA methylation and cellular microenviroment generates long lasting memory effects changing the CSC malignant capacity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4261. doi:1538-7445.AM2012-4261