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

Oncogene volume 35, pages 116–124 (2016)Cite this article

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Abstract

Hepatoma exhibits a series of heterogeneous subpopulations in its cell surface markers, tumorigenicity, invasion and metastatic capability. We previously demonstrated that the CD133−/EpCAM− hepatoma subpopulation was more metastatic than its counterpart; however, the controlling mechanisms are unexplored. The present study aimed to delineate the significance of aberrant hedgehog (Hh) signaling in the mediation of metastases. Fluorescence-activated cell sorting-enriched CD133−/EpCAM− (double negative, DN), Huh-7 cells underwent a transwell selection for metastatic cells (transwell-selected, TS). The TS cells displayed much greater metastatic activity as evidenced by an increased invasion rate, extremely upregulated expression of matrix metalloproteinase (MMP)-1/2/9 genes compared with DN and double-positive (DP) subpopulations. In contrast to DP cells, TS cells lost E-cadherin and were all vimentin-positive as shown by immunocytochemistry. There was a transitional increase in Gli-1/2 gene expression levels from DP, DN to TS subpopulations, which was consistent with elevated Gli-1/2 or Twist-1 protein levels in the nuclear fraction. Furthermore, truncated Gli-1 (tGli-1), which transactivates molecules involved in metastasis, was detected in the highly invasive Huh-7 cell subpopulation, but not in less metastatic hepatoma cells or normal hepatocytes. The enhanced metastatic features with increased expression of MMPs as well as the presence of twist and snail genes in TS Huh-7 cells were reversed by LDE225, a potent Smoothened antagonist. In conclusion, the highly metastatic capability of a unique TS subpopulation was highly attributed to significant epithelial–mesenchymal transition, enhanced Hh activity and aberrant occurrence of a tGli-1 variant, which appears to be responsible for the highly invasive behavior.

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Abbreviations

DAPI:

4', 6-diamidino-2-phenylindole

DP:

double-positive subpopulation (CD133+/EpCAM+)

DN:

double-negative subpopulation (CD133−/EpCAM−)

EMT:

epithelial–mesenchymal transition

FACS:

fluorescence-activation cell sorting

GAPDH:

glyceraldehyde phosphate dehydrogenase

HCC:

hepatocellular carcinoma

Hh:

hedgehog

TS:

CD133−/EpCAM− transwell-selected

MMP:

matrix metalloproteinase

RT–PCR:

reverse transcriptase polymerase chain reaction

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Acknowledgements

This study is partially supported by the Natural Science Foundation of China to Dr Jian Wu (#81272436). Dr Mark A Zern was supported by the California Institute of Regenerative Medicine (#TR0-01857). Ya-Han Fan is the recipient of China Scholarship Council Award (201207610003). The authors are grateful to Bridget McLaughlin for her technical support in the FACS enrichment of hepatoma cell subpopulations at the UC Davis Flow Cytometry Shared Resource, and to Mrs Shu-Hui Sun from Fudan University Shanghai Medical College for her technical assistance in flow cytometric analysis. The abstract of this work was presented in the 64th Annual Meeting of the American Association for the Study of Liver Diseases, Washington, DC, USA, 1–5 November 2013, and published in Hepatology (2013;59:1081A).

Author information

Author notes

  1. Y-H Fan and J Ding: These two authors contribute equally to this work.

Authors and Affiliations

  1. Department of Internal Medicine, Division of Gastroenterology & Hepatology, University of California, Davis Medical Center, Sacramento, CA, USA

    Y-H Fan, S Nguyen, M A Zern & J Wu

  2. Department of Gastroenterology, Xinqiao Hospital, Third Military Medical University, Chongqing, China

    Y-H Fan & S-M Yang

  3. Key Laboratory of Molecular Virology, Fudan University Shanghai Medical College, Shanghai, China

    J Ding, X-J Liu, G Xu, H-Y Zhou, N-N Duan & J Wu

  4. Department of Gastroenterology, Shanghai Jing’an District Central Hospital, Shanghai, China

    J Ding

  5. Institute for Regenerative Cures, University of California, Davis Medical Center, Sacramento, CA, USA

    M A Zern & J Wu

  6. Shanghai Institute of Liver Diseases, Fudan University Affiliated Zhongshan Hospital, Shanghai, China

    J Wu

  7. University of California, Davis Comprehensive Cancer Center, Sacramento, CA, USA

    J Wu

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  1. Y-H Fan
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Correspondence to J Wu.

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Fan, YH., Ding, J., Nguyen, S. et al. Aberrant hedgehog signaling is responsible for the highly invasive behavior of a subpopulation of hepatoma cells. Oncogene 35, 116–124 (2016). https://doi.org/10.1038/onc.2015.67

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