Abstract 2005: Aberrant hedgehog signaling is responsible for the highly invasive behavior of a subpopulation of hepatoma cells

Abstract

Abstract Background: Hepatoma consists of heterogeneous subpopulations in terms of their cell surface markers, tumorigenicity, invasion and metastatic capability. In our previous study, we indicated that the CD133-/EpCAM- hepatoma subpopulation was more metastatic than its counterpart; however the controlling mechanisms are unexplored (J Hepatol 2011;55:838-845). The present study aims to delineate the significance of aberrant hedgehog signaling in the development of metastases. Methods: Huh-7 cells were FACS-enriched into CD133+/EpCAM+ (double positive, DP) and CD133-/EpCAM- (double negative, DN) subpopulations. The double negative cells further underwent Transwell-selection for metastatic cells (Transwell-selected, TS). The metastatic rate, matrix metalloproteinase (MMP) gene expression, epithelial mesenchymal transition (EMT) markers, and hedgehog signaling activities, as well as truncated Gli1 were determined in these subpopulations. Results: TS cells displayed much greater metastatic activity as evidenced by an increased Transwell invasion rate (105 vs. 68.7 and 58.0%, p<0.01), extremely elevated expression of MMP1, 2 and 9 genes (36-3000-fold, p<0.05-0.001) compared to DN and DP populations. There was a nearly 2-fold increase in TGF-β1 gene expression in TS cells compared to DN and DP subpopulations. TS cells lost E-cadherin and were all vimentin-positive as shown by immunocytochemistry in contrast to DP cells. There was a transitional increase in Gli1 gene expression levels from DP, DN to TS subpopulations, which is consistent with elevated Gli-2 and Twist1 levels in the nuclear fraction as detected by Western blot analysis and quantitative RT-PCR. Furthermore, truncated Gli1, which has been indicative of mediating expression of the molecules involved in metastasis, was detected in highly invasive hepatoma cell subpopulations and was the highest in the TS subpopulation; whereas, it did not appear in primary human hepatocytes. Flow cytometric analysis verified that these TS cells maintained a negative cell surface marker profile in their sub-passages. Conclusions: The highly metastatic capability of unique Transwell-selected cells is associated with negative expression of CD133 and EpCAM surface molecules, significant EMT, enhanced hedgehog activity and aberrant expression of truncated Gli1. The appearance of truncated Gli1 is probably responsible for highly invasive behavior of the transwell-selected double-negative subpopulation. Further exploration of molecular mechanisms of abnormal hedgehog signaling in modulating metastases will establish molecular targets for the therapeutic intervention of hepatoma metastases. Citation Format: Yahan Fan, Jia Ding, Jian Wu. Aberrant hedgehog signaling is responsible for the highly invasive behavior of a subpopulation of hepatoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2005. doi:10.1158/1538-7445.AM2014-2005