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Hypoxia Enhances Tumor-Stroma Crosstalk that Drives the Progression of Hepatocellular Carcinoma

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Abstract

Background

Crosstalk between tumor cells and their microenvironment plays a crucial role in the progression of hepatocellular carcinoma (HCC). Hypoxia, a common feature of advanced HCC, has been shown to modulate the evolution of the tumor microenvironment. In this study, we investigated the effect of hypoxia on tumor-stroma crosstalk in HCC.

Methods

Human HCC cell lines (Huh-BAT, SNU-475) were cocultured with an activated human hepatic stellate cell line (HSCs; LX-2) under either normoxic or hypoxic conditions. Cell growth was evaluated with the MTS assay. Apoptotic signaling cascades were assessed by immunoblot analysis. Expression of CD31 and phosphorylated (p-) Akt in HCC tissues was detected by immunohistochemistry.

Results

Coculturing HCC cells with HSCs under hypoxic conditions enhanced their proliferation, migration, and resistance to bile acid (BA)-induced apoptosis compared to coculturing under normoxic conditions. Under hypoxia, of various HSC-derived growth factors, PDGF-BB was the most up-regulated, leading to the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in HCC cells. Immunohistochemical study also revealed that p-Akt was highly expressed in hypoxic, hypovascular HCC as compared to hypervascular HCC. Neutralizing antisera to PDGF-BB or a PI3K inhibitor attenuated the proliferation of HCC cells cocultured with HSCs, and sensitized HCC cells to BA-induced apoptosis, especially under hypoxic conditions.

Conclusions

In conclusion, hypoxic HSC-derived PDGF-BB stimulates the proliferation of HCC cells through activation of the PI3K/Akt pathway, while the inhibition of PDGF-BB or PI3K/Akt pathways enhances apoptotic cell death. Targeting tumor-stroma crosstalk might be a novel therapy in the management of human HCCs.

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Abbreviations

HCC:

Hepatocellular carcinoma

HSC:

Hepatic stellate cell

MTS:

3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium

DAPI:

4′,6-Diamidino-2-phenylindole

BA:

Bile acid

ELISA:

Enzyme-linked immunosorbent assay

RT-PCR:

Reverse transcription-polymerase chain reaction

Akt:

Protein kinase B

PTEN:

Phosphatase and tensin homolog

PI3K:

Phosphoinositide 3-kinase

PDGF:

Platelet-derived growth factor

FGF:

Fibroblast growth factor

TGF:

Transforming growth factor

CTGF:

Connective tissue growth factor

MAPK:

Mitogen-activated protein kinase

OD:

Optical density

SD:

Standard deviation

IHC:

Immunohistochemical

FOXO:

Forkhead transcription factors

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Acknowledgments

This study was supported by a grant from the National R&D Program for Cancer Control, Ministry for Health and Welfare, Republic of Korea (1420050), and by the Liver Research Foundation of Korea.

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    Authors and Affiliations

    1. Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul, 03080, Republic of Korea

      Yuri Cho, Eun Ju Cho, Jeong-Hoon Lee, Su Jong Yu, Yoon Jun Kim, Chung Yong Kim & Jung-Hwan Yoon

    2. Department of Internal Medicine, CHA Gangnam Medical Center, CHA University, Seoul, Republic of Korea

      Yuri Cho

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    1. Yuri Cho
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    2. Eun Ju Cho
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    Correspondence to Jung-Hwan Yoon.

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    Yuri Cho and Eun Ju Cho have contributed equally to this work.

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    Cho, Y., Cho, E.J., Lee, JH. et al. Hypoxia Enhances Tumor-Stroma Crosstalk that Drives the Progression of Hepatocellular Carcinoma. Dig Dis Sci 61, 2568–2577 (2016). https://doi.org/10.1007/s10620-016-4158-6

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    • DOI: https://doi.org/10.1007/s10620-016-4158-6

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