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SOX4 activates CXCL12 in hepatocellular carcinoma cells to modulate endothelial cell migration and angiogenesis in vivo

Oncogene volume 39, pages 4695–4710 (2020)Cite this article

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Abstract

The overexpression of SOX4 in various kinds of cancer cells was associated with poor prognosis for patients. The role of SOX4 in angiogenesis and tumor microenvironment modulation was recently documented in breast cancer but remains unclear in hepatocellular carcinoma (HCC). In our study, the clinical relevance of SOX4 overexpression in HCC and its role in the tumor microenvironment were investigated. The overexpression of SOX4 (SOX4high) in tumor lesions was associated with higher microvessel density (P = 0.012), tumor thrombosis formation (P = 0.012), distant metastasis (P < 0.001), and an independent prognostic factor for disease-free survival in HCC patients (P = 0.048). Endogenous SOX4 knockout in Hep3B cells by the CRISPR/cas9 system reduced the expression of CXCL12, which, in turn, attenuated chemotaxis in human umbilical vein endothelial cells, tube formation in vitro, reduced tumor growth, reticular fiber production, and angiogenesis in vivo in a xenograft mouse model. Treatment with an antagonist targeting CXCR4 (AMD3100), a receptor of CXCL12, inhibited chemotaxis and tube formation in endothelial cells in vitro. The CXCL12 promoter was activated by ectopic expression of a Flag-tagged SOX4 plasmid, endogenous SOX4 knockdown abolished promoter activity of CXCL12 as shown by luciferase assays, and an association with the CXCL12 promoter was identified via chromatin immunoprecipitation in HCC cells. In conclusion, SOX4 modulates the CXCL12 promoter in HCC cells. The secretory CXCL12, in turn, modulates CXCR4 in endothelial cells, reticular fibers to regulate the tumor microenvironment and modulate neovascularization, which might contribute to the distant metastasis of tumors.

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Fig. 1: The overexpression of SOX4 was associated with poor prognosis in patients with hepatocellular carcinoma (HCC).
Fig. 2: Knockout of endogenous expression of SOX4 in hepatocellular carcinoma (HCC) cell lines via the CRISPR/cas9 system attenuated chemotaxis of endothelial cells and reduced expression of CXCL12.
Fig. 3: SOX4 regulated the CXCL12 promoter.
Fig. 4: The chemotaxis of endothelial cells was impaired when SOX4 or CXCL12 was knocked down in Hep3B cells.
Fig. 5: SOX4 modulates tube formation through the CXCR4-CXCL12 axis.
Fig. 6: Tumor growth in vivo was impaired when endogenous SOX4 expression was knocked out.
Fig. 7: H&E, trichrome, reticulin, SOX4, and CD34 staining in tumor sections were derived from Hep3B or Hep3B SOX4−/− cells.

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Acknowledgements

The authors thank all the members of the Cancer Center, Genomic Medicine Core Laboratory, and Center for Advanced Molecular Imaging and Translation, Chang Gung Memorial Hospital, Linkou, for their invaluable help. We are also grateful to Jang-Hau Lian and Yi-Ping Liu for their assistance in data retrieval and processing.

Funding

Financial support by Chang Gung Medical Foundation in Taiwan: CMRPD1G0611 and CMRPD1H0661 for Dr CN Tsai, CORPG1G0021 and CMRPG1J0061 for Dr MC Yu and Ministry of Science and Technology: MOST 103-2314-B-182A-082-MY1-3 and MOST 106-2314-B-182A-113 for Dr MC Yu.

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Authors and Affiliations

  1. Graduate Institute of Clinical Medical Sciences, Chang-Gung University, Guishan Dist, Taoyuan City, 33302, Taiwan

    Chi-Neu Tsai, Chao-Wei Lee & Jong-Hwei Su Pang

  2. Department of Surgery, Chang Gung Memorial Hospital, Linkou Branch, Guishan Dist, Taoyuan City, 33305, Taiwan

    Chi-Neu Tsai, Shu-Chuan Yu, Chao-Wei Lee, Chun-Hsin Wu & Ming-Chin Yu

  3. Department of Pathology, Wan Fang Hospital, Wenshan Dist, Taipei City, 11696, Taiwan

    Sey-En Lin

  4. Department of Pathology, School of Medicine, College of Medicine, Taipei Medical University, Taipei City, 11031, Taiwan

    Sey-En Lin

  5. Center for Advanced Molecular Imaging and Translation (CAMIT), Chang Gung Memorial Hospital, Taoyuan City, 33305, Taiwan

    Yi-Hsiu Chung

  6. Genomic Medicine Core Laboratory, Chang Gung Memorial Hospital, Linkou Branch, Guishan Dist, Taoyuan City, 33305, Taiwan

    Chia-Lung Tsai

  7. Department of Gastroenterology and Hepatology, Chang Gung Memorial Hospital, Linkou Branch, Taoyuan City, 33305, Taiwan

    Sen-Yung Hsieh

  8. Department of Surgery, Xiamen Chang Gung Hospital, Xiamen, 361028, China

    Ming-Chin Yu

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Tsai, CN., Yu, SC., Lee, CW. et al. SOX4 activates CXCL12 in hepatocellular carcinoma cells to modulate endothelial cell migration and angiogenesis in vivo. Oncogene 39, 4695–4710 (2020). https://doi.org/10.1038/s41388-020-1319-z

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