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Systematic Analysis of Cytostatic TGF-Beta Response in Mesenchymal-Like Hepatocellular Carcinoma Cell Lines

Abstract

Background

Hepatocellular carcinoma (HCC) is one of the most challenging malignancies, with high morbidity and mortality rates. The transforming growth factor-β (TGF-β) pathway plays a dual role in HCC, acting as both tumor suppressor and promoter. A thorough understanding of the mechanisms underlying its opposing functions is important. The growth suppressive effects of TGF-β remain largely unknown for mesenchymal HCC cells. Using a systematic approach, here we assess the cytostatic TGF-β responses and intracellular transduction of the canonical TGF-β/Smad signaling cascade in mesenchymal-like HCC cell lines.

Methods

Nine mesenchymal-like HCC cell lines, including SNU182, SNU387, SNU398, SNU423, SNU449, SNU475, Mahlavu, Focus, and Sk-Hep1, were used in this study. The cytostatic effects of TGF-β were evaluated by cell cycle analysis, BrdU labeling, and SA-β-Gal assay. RT-PCR and western blot analysis were utilized to determine the mRNA and protein expression levels of TGF-β signaling components and cytostatic genes. Immunoperoxidase staining and luciferase reporter assays were performed to comprehend the transduction of the canonical TGF-β pathway.

Results

We report that mesenchymal-like HCC cell lines are resistant to TGF-β-induced growth suppression. The vast majority of cell lines have an active canonical signaling from the cell membrane to the nucleus. Three cell lines had lost the expression of cytostatic effector genes.

Conclusion

Our findings reveal that cytostatic TGF-β responses have been selectively lost in mesenchymal-like HCC cell lines. Notably, their lack of responsiveness was not associated with a widespread impairment of TGF-β signaling cascade. These cell lines may serve as valuable models for studying the molecular mechanisms underlying the loss of TGF-β-mediated cytostasis during hepatocarcinogenesis.

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Acknowledgements

Serif Senturk is a recipient of the Young Scientists Award Program of the Turkish Academy of Sciences (TUBA GEBIP 2017) and the Science Academy’s Young Scientist Awards Program (BAGEP 2019). The schematic for the canonical TGF-β signaling cascade was created with BioRender.com. pSBE4-Luc was a gift from Bert Vogelstein (Addgene plasmid # 16495) and p3TP-Lux was a gift from Joan Massague (Addgene plasmid # 11767).

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Contributions

MZG and MU performed cell cycle experiments as well as BrdU incorporation assay and analyzed the data. The remaining experiments were carried out by SS. SS and MO conceived and designed the study and analyzed the data. MZG, MU, and SS all made substantial contributions to the preparation of the figures and drafting the manuscript. The final manuscript was read and approved by all authors.

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Correspondence to Serif Senturk.

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Gungor, M.Z., Uysal, M., Ozturk, M. et al. Systematic Analysis of Cytostatic TGF-Beta Response in Mesenchymal-Like Hepatocellular Carcinoma Cell Lines. J Gastrointest Canc 52, 1320–1335 (2021). https://doi.org/10.1007/s12029-021-00704-z

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Keywords

  • Hepatocellular carcinoma
  • TGF-β pathway
  • Cytostatic response
  • HCC cell lines
  • Mesenchymal