P0229 : Jagged2-mediated notch signaling regulates cellular plasticity and promotes a cancer stem cell phenotype in HCC

Abstract

Background and Aims: Cancer cells within tumors can exist in various states of differentiation and can flux between these states, a phenomenonmediated by epigenetic changes. Determining epigenetic changes is critical for effective therapy because dedifferentiation can drive a cancer stem cell (CSC) phenotype with more aggressive behaviour that is resistant to current therapies. Notch signalling is a major mediator of cell fate and is a critical signalling pathway in maintaining the cells in an undifferentiated state. Our study characterises the role Notch signalling plays in maintaining a LCSC phenotype. Methods: Undifferentiated cells with a liver cancer stem cell (LCSC) phenotype were isolated from HCC cell lines HuH7 and PLC/PRF/5 using flow cytometry based on the expression of the classical CSC marker Oct4. The expression of Notch signalling components were examined in these undifferentiated cells by Western blot. Tumour sphere assays and cell proliferation were undertaken on undifferentiated cells treated with recombinant human Jagged2 (rhJagged2) and siRNA specifically targeting Jagged2 (siRNA-Jagged2). The expression of transcription factors involved in dedifferentiation was then determined in the HCC cell lines. The expression of Jagged2 in tumor and adjacent non-tumor tissue from HCC patients was evaluated by Western blot. Results: Increased expression of Jagged2 and the Notch functional receptor subunit NICD1 was observed in Oct4+ LCSCs. Treatment of LCSCs with rhJagged2 resulted in activation of Notch signalling as manifested by increased expression of Notch signalling components, particularly the Notch downstream targets Hey1 and NICD1. On the other hand, inhibition of Jagged2 by siRNA led to a significant reduction in the expression of Notch signalling components. rhJagged2-treated LCSCs showed increased cell proliferation and tumor sphere formation which were significantly blunted by siRNA-Jagged2. HCC cells PLC/PRF/5 and HuH7 treated with rhJagged2 resulted in a significant up-regulation of Sox-2. Increased expression of Jagged2 was also observed in human HCC tumour tissues compared to adjacent non-tumour liver. Conclusions: Jagged2 is likely an important molecule in the maintenance of CSC phenotype and differentiation status; this in turn is involved in HCC development. Up-regulation of Jagged2 in HCC tissues suggests that Jagged2 targeting may be a therapeutic option for HCC. Studies are currently underway to explore the therapeutic benefit of targeting Jagged2 in HCC.