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Tumor–matrix interaction induces phenotypic switching in liver cancer cells

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Abstract

Background

Intrahepatic cholangiocarcinoma (ICC) is characterized by fibrous stroma and clinical behavior more aggressive than that of hepatocellular carcinoma (HCC). Scirrhous HCC is a subtype of HCC with fibrous stroma, frequently has partial cholangiocytic differentiation, and is more likely to have an aggressive behavior. This study explored the interaction of liver cancer cells with the extracellular matrix.

Methods and results

Liver cancer cells grown on collagen 1-coated plates showed upregulation of cholangiocytic marker expression but downregulation of hepatocytic marker expression. Three-dimensional sphere culture and Boyden chamber assay showed enhanced invasion and migration ability in collagen 1-conditioned liver cancer cells. Interaction with collagen 1 reduced liver cancer cell proliferation. RNA sequencing showed that in the liver cancer cells, collagen 1 upregulated cell cycle inhibitor expression and cell–matrix interaction, tumor migration, and angiogenesis pathways, but downregulated liver metabolic function pathways. Cholangiocytic differentiation and invasiveness induced by collagen 1 was mediated by the mitogen-activated protein kinase (MAPK) pathway, which was regulated by cell–matrix interaction-induced Src activation. Analysis of the Cancer Genome Atlas cohort showed that collagen 1 induced and suppressed genes were highly enriched in ICC and HCC, respectively. In HCC samples, collagen 1-regulated genes were strongly coexpressed and correlated with COL1A1 expression.

Conclusions

Liver cancer cell–matrix interaction induces cholangiocytic differentiation and switches liver cancer cells from a proliferative to an invasive phenotype through the Src/MAPK pathway, which may partly explain the differences in the behaviors of HCC and ICC.

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Data availability

The RNA-seq data were deposited in NCBI Genome Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/) under the accession number: GSE176270. Other data are available on request.

Abbreviations

3D:

Three-dimensional

AFP:

α-Fetoprotein

ALB:

Albumin

ATAC-seq:

Assay of transposase-accessible chromatin using sequencing

DMEM:

Dulbecco’s modified Eagle’s medium

ECM:

Extracellular matrix

FAK:

Focal adhesion kinase

GESA:

Gene set enrichment analysis

HNF-1β:

Hepatocyte nuclear factor 1β

ICC:

Intrahepatic cholangiocarcinoma

KRT19:

Keratin 19

MAPK:

Mitogen-activated protein kinase

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H- tetrazoliumbromide

ORA:

Over-representative analysis

PAK1:

P21-activated kinase 1

PBS:

Phosphate buffered saline

RNA-seq:

RNA sequencing

RT-PCR:

Reverse transcriptase-polymerase chain reaction

TCGA:

The Cancer Genome Atlas

TF:

Transferrin

TSS:

Transcriptional start sites

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Acknowledgements

We thank the 2nd and 3rd Core Laboratory of National Taiwan University Hospital and Translational core facility of Taipei Medical University for technical support.

Funding

The study was supported by grants from Ministry of Science and Technology, Republic of China (grant number: 108-2320-B-002-059-MY3 and 109-2314-B-002-082).

Author information

Author notes

  1. Ray-Hwang Yuan and Chia-Lang Hsu contributed equally to this work.

Authors and Affiliations

  1. Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan

    Ray-Hwang Yuan

  2. Department of Surgery, Hsinchu Branch, National Taiwan University Hospital, Hsinchu, Taiwan

    Ray-Hwang Yuan

  3. Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan

    Chia-Lang Hsu

  4. Graduate Institute of Pathology, National Taiwan University, Taipei, Taiwan

    Yu-Lin Jhuang & Yung-Ming Jeng

  5. Department of Pathology, National Taiwan University Hospital, Taipei, Taiwan

    Yu-Lin Jhuang & Yung-Ming Jeng

  6. Joint Biobank Office of Human Research, Taipei Medical University, Taipei, Taiwan

    Yun-Ru Liu & Tsung-Han Hsieh

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  1. Ray-Hwang Yuan
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Contributions

Study concept and design: JYM and YRH; methodology and technical support: YRH and JYL; analysis and interpretation of data: HCL, LYR and HTH; writing, review and/or revision of the manuscript: JYM, YRH, and HCL.

Corresponding author

Correspondence to Yung-Ming Jeng.

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Conflict of interest

Ray-Hwang Yuan, Chia-Lang Hsu, Yu-Lin Jhuang, Yun-Ru Liu, Tsung-Han Hsieh, and Yung-Ming Jeng have no conflicts of interest relevant to this article.

Ethic approval

This study was approved under the regulations of the Research Ethics Committee of the National Taiwan University Hospital (approval number: 201911077RINB) and conducted according to the principles of the Declaration of Helsinki.

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Supplementary Information

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Supplementary file1 (DOCX 16 KB)

Supplementary file2 (DOCX 22 KB)

Supplementary file3 (DOCX 31 KB)

Supplementary file4 (DOCX 20 KB)

Supplementary Fig. 1.

Supplementary file5 KRT19 and vimentin expression in liver cancer cell lines. HepJ5, HCC36, HA22T, and Mahlava cells express vimentin. HA59T and HCC36 cells have intrinsically high KRT19 expression. Hep3B and PLC5 cells lack KRT19 and vimentin expression. Huh7 and HepG2 have low but detectable KRT19 expression but no vimentin expression. (TIF 244 KB)

Supplementary Fig. 2.

Supplementary file6The effects of interaction with collagen 1 or fibronectin on the expression levels of hepatocellular or cholangiocytic differentiation markers in Hep3B, PLC5, HCC36, and HA59T cells. N.D.: not detected (Ct value >30). (TIF 591 KB)

Supplementary Fig. 3.

Supplementary file7Morphology of Huh7 and HepG2 cells on non-coated and collagen 1–coated plates. Huh7 cells grown on non-coated and collagen 1–coated plates have similar morphology. HepG2 cells forms tightly adherent tumor nests on non-coated plates, but they spread out as single discohesive cells on the collagen 1–coated plate. (TIF 2146 KB)

Supplementary Fig. 4.

Supplementary file8The Notch and transforming growth factor-β pathways are not involved in collagen 1–induced KRT19 expression in Huh7 and HepG2 cells. Treatment with the γ-secretase inhibitor DAPT (a) or SMAD2/3 inhibitor SB4315542 (b) has no effect on collagen 1–induced upregulation of KRT19 expression. (TIF 378 KB)

Supplementary Fig. 5.

Supplementary file9a Three-dimensional spheroid culture assay showing that HepG2 cells have an invasive morphology when grown in Matrigel supplemented with collagen 1. Treatment with MEK inhibitor PD98059 (20 µM) or Src inhibitor dasatinib (100 nM) returns the morphology to a noninvasive spheroid. b, c HepG2 cells grown on collagen 1 have elevated expression of the cholangiocytic marker KRT19 (b) and invasiveness genes (c) but reduced expression of the hepatocytic marker ALB (b). PD98059 treatment abolishes the changes in gene expression. *P < 0.05, **P < 0.01, ***P < 0.001. (TIF 456 KB)

Supplementary Fig. 6.

Supplementary file10Huh7 (a, b) and HepG2 (c, d) cells grown on collagen 1 have elevated expression levels of the cholangiocytic marker KRT19 (a, c) and invasiveness genes (b) but reduced expression levels of the hepatocytic marker ALB (a, c). Dasatinib treatment (100 nM) abolishes the changes in gene expression. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (TIF 383 KB)

Supplementary Fig. 7.

Supplementary file11Fragment size, the location of ATAC peaks related to transcription start site, and the distribution of ATAC peaks. a Huh7 cells grown on a non-coated plate, b Huh7 cells grown on a collagen 1–coated plate, c HepG2 cells grown on a non-coated plate, d HepG2 cells grown on a collagen 1–coated plate. (TIF 426 KB)

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Yuan, RH., Hsu, CL., Jhuang, YL. et al. Tumor–matrix interaction induces phenotypic switching in liver cancer cells. Hepatol Int 16, 562–576 (2022). https://doi.org/10.1007/s12072-022-10315-w

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