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

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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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References

  1. Globocan. “WHO Liver Cancer Fact Sheet”. 2020.

  2. Llovet JM et al. “Hepatocellular carcinoma,” Nat Rev Dis Primer. 2021;7(1). https://doi.org/10.1038/s41572-020-00240-3.

  3. Villanueva A. “Hepatocellular carcinoma,” N Engl J Med. 2019;1450–1462. https://doi.org/10.1056/NEJMra1713263.

  4. Llovet JM, Zucman-rossi J, Pikarsky E, Sangro B, Sherman M, Gores G. “Hepatocellular carcinoma,” Nat. Rev. Dis. Primer. 2016;2. https://doi.org/10.1038/nrdp.2016.18.

  5. Zucman-rossi J, Villanueva A, Nault J, Llovet JM. Genetic landscape and biomarkers of hepatocellular carcinoma. YGAST. 2015;149(5):1226-1239.e4. https://doi.org/10.1053/j.gastro.2015.05.061.

    Article  CAS  Google Scholar 

  6. Ozturk M, Arslan-Ergul A, Bagislar S, Senturk S, Yuzugullu H. Senescence and immortality in hepatocellular carcinoma. Cancer Lett. 2009;286(1):103–13. https://doi.org/10.1016/j.canlet.2008.10.048.

    Article  CAS  PubMed  Google Scholar 

  7. Yuzugullu H et al. Canonical Wnt signaling is antagonized by noncanonical Wnt5a in hepatocellular carcinoma cells. Mol Cancer. 2009;8:1–20. https://doi.org/10.1186/1476-4598-8-90.

    Article  CAS  Google Scholar 

  8. Caruso S et al. “Analysis of liver cancer cell lines identifies agents with likely efficacy against hepatocellular carcinoma and markers of response,” Gastroenterology. 2019;1–17. https://doi.org/10.1053/j.gastro.2019.05.001.

  9. Dituri F, Mancarella S, Cigliano A, Chieti A, Giannelli G. TGF-β as multifaceted orchestrator in HCC progression: signaling, EMT, immune microenvironment, and novel therapeutic perspectives. Semin Liver Dis. 2019;39(1):53–69. https://doi.org/10.1055/s-0038-1676121.

    Article  CAS  PubMed  Google Scholar 

  10. Weiss A, Attisano L. The TGFbeta superfamily signaling pathway. WIREs Dev Biol. 2013;2:47–63. https://doi.org/10.1002/wdev.86.

    Article  CAS  Google Scholar 

  11. Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-β family signalling. Nature. 2003;425:577–84.

    Article  CAS  Google Scholar 

  12. Zhang YE “Non-Smad signaling pathways of the TGF- b family,” Cold Spring Harb Perspect Biol. 2017;1–18.

  13. Massagué J, Seoane J, Wotton D. Smad transcription factors. Genes Dev. 2005;19(23):2783–810. https://doi.org/10.1101/gad.1350705.

    Article  CAS  PubMed  Google Scholar 

  14. Arrese M, Cabrera D, Hernandez A, Astete L, Estrada L. “TGF- β and hepatocellular carcinoma : when a friend becomes an enemy,” Curr Protein Pept Sci. 2018;19. https://doi.org/10.2174/1389203718666171117112619.

  15. Seoane J, Gomis RR. “TGF- b family signaling in tumor suppression and cancer progression,” Cold Spring Harb Perspect Biol. 2017;9. https://doi.org/10.1101/cshperspect.a022277.

  16. Dzieran J et al. Comparative analysis of TGF-β / Smad signaling dependent cytostasis in human hepatocellular carcinoma cell lines. PLoS One. 2013;8(8):1–18. https://doi.org/10.1371/journal.pone.0072252.

    Article  CAS  Google Scholar 

  17. Hao Y, Baker D, Ten Dijke P. “TGF-β-mediated epithelial-mesenchymal transition and cancer metastasis,” Int J Mol Sci. 2019;20(11). https://doi.org/10.3390/ijms20112767.

  18. Fabregat I, Caballero-Díaz D. “Transforming growth factor-β-induced cell plasticity in liver fibrosis and hepatocarcinogenesis,” Front Oncol. 2018;8. https://doi.org/10.3389/fonc.2018.00357.

  19. Senturk S, Mumcuoglu M, Gursoy-Yuzugullu O, Cingoz B, Akcali KC, Ozturk M. Transforming growth factor-beta induces senescence in hepatocellular carcinoma cells and inhibits tumor growth. Hepatology. 2010;52(3):966–74. https://doi.org/10.1002/hep.23769.

    Article  CAS  PubMed  Google Scholar 

  20. Coulouarn C, Factor VM, Thorgeirsson SS. Transforming growth factor-β gene expression signature in mouse hepatocytes predicts clinical outcome in human cancer. Hepatology. 2008;47(6):2059–67. https://doi.org/10.1038/jid.2014.371.

    Article  CAS  PubMed  Google Scholar 

  21. Huh N, Utakoji T. Production of HBs-antigen by two new human hepatoma cell lines and its enhancement by dexamethasone. Gan. 1981;72(1):178–9.

    CAS  PubMed  Google Scholar 

  22. Park JG et al. Characterization of cell lines established from human hepatocellular carcinoma. Int J Cancer. 1995;62(3):276–82. https://doi.org/10.1002/ijc.2910620308.

    Article  CAS  PubMed  Google Scholar 

  23. Hirschfield H et al. In vitro modeling of hepatocellular carcinoma molecular subtypes for anti-cancer drug assessment. Exp Mol Med. 2018;50(1): e419. https://doi.org/10.1038/emm.2017.164.

    Article  PubMed  PubMed Central  Google Scholar 

  24. Heffelfinger SC, Hawkins HH, Barrish J, Taylor L, Darlington GJ. “SK HEP-1: a human cell line of endothelial origin”, Vitro Cell. Dev Biol J Tissue Cult Assoc. 1992;28A(2):136–42. https://doi.org/10.1007/BF02631017.

    Article  CAS  Google Scholar 

  25. Alexander JJ. “Human hepatoma cell lines,” in Neoplasms of the liver, K. Okuda and K. G. Ishak, Eds. Tokyo: Springer Japan. 1987;47–56. https://doi.org/10.1007/978-4-431-68349-0_4.

  26. He L, Isselbacher KJ, Wands JR, Goodman HM, Shih C, Quaroni A. Establishment and characterization of a new human hepatocellular carcinoma cell line. Vitro. 1984;20(6):493–504. https://doi.org/10.1007/BF02619623.

    Article  CAS  Google Scholar 

  27. Inagaki M, Moustakas A, Lin HY, Lodish HF, Carr BI. Growth inhibition by transforming growth factor beta (TGF-beta) type I is restored in TGF-beta-resistant hepatoma cells after expression of TGF-beta receptor type II cDNA. Proc Natl Acad Sci USA. 1993;90(11):5359–63. https://doi.org/10.1073/pnas.90.11.5359.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Cerami E et al. The cBio Cancer Genomics Portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov. 2012;2(5):401–4. https://doi.org/10.1158/2159-8290.CD-12-0095.

    Article  Google Scholar 

  29. Tate JG et al. COSMIC: the Catalogue Of Somatic Mutations In Cancer. Nucleic Acids Res. 2019;47(D1):D941–7. https://doi.org/10.1093/nar/gky1015.

    Article  CAS  PubMed  Google Scholar 

  30. Barretina J et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature. 2012;483(7391):603–7. https://doi.org/10.1038/nature11003.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Kitisin K et al. Disruption of transforming growth factor- b signaling through b -spectrin ELF leads to hepatocellular cancer through cyclin D1 activation. Oncogene. 2007;26:7103–10. https://doi.org/10.1038/sj.onc.1210513.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Baek MJ et al. “p16 is a major inactivation target in hepatocellular carcinoma”, Cancer no. Cdk. 2000;4:60–8.

    Google Scholar 

  33. Liew CT et al. High frequency of p16 INK4A gene alterations in hepatocellular carcinoma. Oncogene. 1999;18:789–95.

    Article  CAS  Google Scholar 

  34. Tannapfel A et al. INK4a-ARF alterations and p53 mutations in hepatocellular carcinomas. Oncogene. 2001;20:7104–9.

    Article  CAS  Google Scholar 

  35. Zang J et al. P16 gene hypermethylation and hepatocellular carcinoma : a systematic review and meta-analysis. World J Gastroenterol. 2011;17(25):3043–8. https://doi.org/10.3748/wjg.v17.i25.3043.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Yarchoan M et al. “Recent developments and therapeutic strategies against hepatocellular carcinoma”. 2019;79(17):4326–4331. https://doi.org/10.1158/0008-5472.CAN-19-0803.

  37. Nault JC et al. Clinical impact of genomic diversity from early to advanced hepatocellular carcinoma. Hepatology. 2019;71:164–82. https://doi.org/10.1002/hep.30811.

    Article  CAS  PubMed  Google Scholar 

  38. Nagaraj NS, Datta PK. Targeting the transforming growth factor-β signaling pathway in human cancer. Expert Opin Investig Drugs. 2010;19(1):77–91. https://doi.org/10.1517/13543780903382609.

    Article  CAS  PubMed  Google Scholar 

  39. Haque S, Morris JC. Transforming growth factor-β: a therapeutic target for cancer. Hum Vaccines Immunother. 2017;13(8):1741–50. https://doi.org/10.1080/21645515.2017.1327107.

    Article  Google Scholar 

  40. Caja L et al “Differential intracellular signalling induced by TGF- β in rat adult hepatocytes and hepatoma cells : implications in liver carcinogenesis”. 2007;19:683–694. https://doi.org/10.1016/j.cellsig.2006.09.002.

  41. Sanchez A, Alberto MA, Benito M, Fabregat I. Apoptosis induced by transforming growth factor-β in fetal hepatocyte primary cultures. J Biol Chem. 1996;271(13):7416–22. https://doi.org/10.1074/jbc.271.13.7416.

    Article  CAS  PubMed  Google Scholar 

  42. Kim DH, Kim WD, Kim SK, Moon DH, Lee SJ. “TGF-β1-mediated repression of SLC7A11 drives vulnerability to GPX4 inhibition in hepatocellular carcinoma cells,” Cell Death Dis. 2007;11(5). https://doi.org/10.1038/s41419-020-2618-6.

  43. Jong H et al. Attenuation of transforming growth factor β-induced growth inhibition in human hepatocellular carcinoma cell lines by cyclin D1 overexpression. Biochem Biophys Res Commun. 2002;292:383–9. https://doi.org/10.1006/bbrc.2002.6666.

    Article  CAS  PubMed  Google Scholar 

  44. Yakicier MC, Irmak MB, Romano A, Kew M, Ozturk M. Smad2 and Smad4 gene mutations in hepatocellular carcinoma. Oncogene. 1999;18:4879–83. https://doi.org/10.1038/sj.onc.1202866.

    Article  CAS  PubMed  Google Scholar 

  45. Di Guglielmo GM, Le Roy C, Goodfellow AF, Wrana JL. “Distinct endocytic pathways regulate TGF- β receptor signalling and turnover”. Nat Cell Biol. 2003;5. https://doi.org/10.1038/ncb975.

  46. Mishra B et al. “Loss of cooperative function of transforming growth factor- b signaling proteins, smad3 with embryonic liver fodrin, a b -spectrin, in primary biliary cirrhosis”. 2004;(5)637–645. https://doi.org/10.1111/j.1478-3231.2004.0958.x.

  47. Dijke P, Miyazono K, Heldin C. Signaling inputs converge on nuclear effectors in TGF-β signaling. Trends Biochem Sci. 2000;25(2):64–70. https://doi.org/10.1016/S0968-0004(99)01519-4.

    Article  PubMed  Google Scholar 

  48. Vidakovic AT et al. Context-specific effects of TGF-β/SMAD3 in cancer are modulated by the epigenome. Cell Rep. 2015;13:2480–90. https://doi.org/10.1016/j.celrep.2015.11.040.

    Article  CAS  Google Scholar 

  49. Hill CS. “Transcriptional control by the SMADs,” Cold Spring Harb Perspect Biol. 2016;8. https://doi.org/10.1101/cshperspect.a022079.

  50. Kojima T et al. Transforming growth factor-β induces epithelial to mesenchymal transition by down-regulation of claudin-1expression and the fence function in adult rat hepatocytes. Liver Int. 2008;28(4):534–45. https://doi.org/10.1111/j.1478-3231.2007.01631.x.

    Article  CAS  PubMed  Google Scholar 

  51. Ohashi S et al. Epidermal growth factor receptor and mutant p53 expand an esophageal cellular subpopulation capable of epithelial-to-mesenchymal transition through ZEB transcription factors. Cancer Res. 2010;70(10):4174–85. https://doi.org/10.1158/0008-5472.CAN-09-4614.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Postigo AA. Opposing functions of ZEB proteins in the regulation of the TGFβ/BMP signaling pathway. EMBO J. 2003;22(10):2443–52. https://doi.org/10.1093/emboj/cdg225.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Acun T, Oztas E, Yagci T, Yakicier MC. SIP1 is downregulated in hepatocellular carcinoma by promoter hypermethylation. BMC Cancer. 2011;11(1):223. https://doi.org/10.1186/1471-2407-11-223.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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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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Author notes

  1. Medine Zeynep Gungor and Merve Uysal contributed equally to this work.

Authors and Affiliations

  1. Izmir Biomedicine and Genome Center, Izmir, 35340, Turkey

    Medine Zeynep Gungor, Merve Uysal, Mehmet Ozturk & Serif Senturk

  2. Department of Genome Sciences and Molecular Biotechnology, Izmir International Biomedicine and Genome Institute, Dokuz Eylul University, Izmir, 35340, Turkey

    Medine Zeynep Gungor, Merve Uysal & Serif Senturk

  3. Department of Medical Biology, Faculty of Medicine, Izmir Tinaztepe University, Izmir, 35400, Turkey

    Mehmet Ozturk

  4. Department of Molecular Biology and Genetics, Bilkent University, Ankara, 06800, Turkey

    Mehmet Ozturk & Serif Senturk

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  1. Medine Zeynep Gungor
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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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