Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Loss of KLF4 and consequential downregulation of Smad7 exacerbate oncogenic TGF-β signaling in and promote progression of hepatocellular carcinoma

Oncogene volume 36, pages 2957–2968 (2017)Cite this article

Subjects

Abstract

Hyperactivation of transforming growth factor-β (TGF-β) signaling pathway is a common feature of hepatocellular carcinoma (HCC) progression. However, the driver factors leading to enhanced TGF-β activity are not well characterized. Here, we explore the mechanisms that loss of Krüppel-like factor 4 (KLF4) exacerbates oncogenic TGF-β signaling in human HCC. The expression of KLF4 and TGF-β signaling components in primary HCC and their clinicopathologic relevance and significance was evaluated by using tissue microarray and immunohistochemistry. Cellular and molecular impacts of altered KLF4 expression and TGF-β signaling were determined using immunofluorescence, western blot, reverse-transcriptase PCR, chromatin immunoprecipitation and promoter reporter assays. Loss of KLF4 expression in primary HCC closely correlated with decreased Smad7 expression, increased p-Smad2/3 expression and independently predicts reduced overall and relapse-free survival after surgery. TGF-β signaling components were expressed in most HCC cells, and activation of TGF-β signaling promoted cell migration and invasion. Enforced KLF4 expression blocked TGF-β signal transduction and inhibited cell migration and invasion via activation of Smad7 transcription, whereas deletion of its C-terminal zinc-finger domain diminished this effect. KLF4 protein physically interacts with the Smad7 promoter. Promoter deletion and point mutation analyses revealed that a region between nucleotides –15 bp and –9 bp of the Smad7 promoter was required for the induction of Smad7 promoter activity by KLF4. Our data indicate that KLF4 suppresses oncogenic TGF-β signaling by activation of Smad7 transcription, and that loss of KLF4 expression in primary HCC may contribute to activation of oncogenic TGF-β signaling and subsequent tumor progression.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

Similar content being viewed by others

Abbreviations

ChIP:

chromatin immunoprecipitation

FFPE:

formalin-fixed paraffin-embedded

KLF4:

Krüppel-like factor 4

EMT:

epithelial-to-mesenchymal transition

ZDF:

zinc-finger domain

PBS:

phosphate-buffered saline

PBS-T:

PBS containing 0.1% Triton X-100

PCR:

polymerase chain reaction

qPCR:

quantitative PCR

RT:

reverse transcription

siRNA:

small interfering RNA

TGF:

transforming growth factor

TMA:

tissue microarray

UTR:

untranslated region.

References

  1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D . Global cancer statistics. CA Cancer J Clin 2011; 61: 69–90.

    Article  Google Scholar 

  2. Mazzocca A, Antonaci S, Giannelli G . The TGF-beta signaling pathway as a pharmacological target in a hepatocellular carcinoma. Curr Pharm Des 2012; 18: 4148–4154.

    Article  CAS  Google Scholar 

  3. Meindl-Beinker NM, Matsuzaki K, Dooley S . TGF-beta signaling in onset and progression of hepatocellular carcinoma. Dig Dis 2012; 30: 514–523.

    Article  Google Scholar 

  4. Yan X, Liu Z, Chen Y . Regulation of TGF-beta signaling by Smad7. Acta Biochim Biophys Sin (Shanghai) 2009; 41: 263–272.

    Article  CAS  Google Scholar 

  5. Baek HJ, Pishvaian MJ, Tang Y, Kim TH, Yang S, Zouhairi ME et al. Transforming growth factor-beta adaptor, beta2-spectrin, modulates cyclin dependent kinase 4 to reduce development of hepatocellular cancer. Hepatology 2011; 53: 1676–1684.

    Article  CAS  Google Scholar 

  6. 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: 966–974.

    Article  CAS  Google Scholar 

  7. Zhu H, Wu K, Yan W, Hu L, Yuan J, Dong Y et al. Epigenetic silencing of DACH1 induces loss of transforming growth factor-beta1 antiproliferative response in human hepatocellular carcinoma. Hepatology 2013; 58: 2012–2022.

    Article  CAS  Google Scholar 

  8. Reichl P, Haider C, Grubinger M, Mikulits W . TGF-beta in epithelial to mesenchymal transition and metastasis of liver carcinoma. Curr Pharm Des 2012; 18: 4135–4147.

    Article  CAS  Google Scholar 

  9. Fransvea E, Mazzocca A, Antonaci S, Giannelli G . Targeting transforming growth factor (TGF)-betaRI inhibits activation of beta1 integrin and blocks vascular invasion in hepatocellular carcinoma. Hepatology 2009; 49: 839–850.

    Article  CAS  Google Scholar 

  10. Mazzocca A, Fransvea E, Lavezzari G, Antonaci S, Giannelli G . Inhibition of transforming growth factor beta receptor I kinase blocks hepatocellular carcinoma growth through neo-angiogenesis regulation. Hepatology 2009; 50: 1140–1151.

    Article  CAS  Google Scholar 

  11. Mazzocca A, Fransvea E, Dituri F, Lupo L, Antonaci S, Giannelli G . Down-regulation of connective tissue growth factor by inhibition of transforming growth factor beta blocks the tumor-stroma cross-talk and tumor progression in hepatocellular carcinoma. Hepatology 2010; 51: 523–534.

    Article  CAS  Google Scholar 

  12. Lin TH, Shao YY, Chan SY, Huang CY, Hsu CH, Cheng AL . High serum transforming growth factor-beta1 levels predict outcome in hepatocellular carcinoma patients treated with sorafenib. Clin Cancer Res 2015; 21: 3678–3684.

    Article  CAS  Google Scholar 

  13. Giannelli G, Villa E, Lahn M . Transforming growth factor-beta as a therapeutic target in hepatocellular carcinoma. Cancer Res 2014; 74: 1890–1894.

    Article  CAS  Google Scholar 

  14. Wei D, Wang L, Yan Y, Jia Z, Gagea M, Li Z et al. KLF4 is essential for induction of cellular Identity change and acinar-to-ductal reprogramming during early pancreatic carcinogenesis. Cancer Cell 2016; 29: 324–338.

    Article  CAS  Google Scholar 

  15. Zhang Y, Wang Y, Liu Y, Wang N, Qi Y, Du J . Kruppel-like factor 4 transcriptionally regulates TGF-beta1 and contributes to cardiac myofibroblast differentiation. PLoS One 2013; 8: e63424.

    Article  CAS  Google Scholar 

  16. Feinberg MW, Cao Z, Wara AK, Lebedeva MA, Senbanerjee S, Jain MK . Kruppel-like factor 4 is a mediator of proinflammatory signaling in macrophages. J Biol Chem 2005; 280: 38247–38258.

    Article  CAS  Google Scholar 

  17. Ding B, Liu P, Liu W, Sun P, Wang CL . Emerging roles of kruppel-like factor 4 in cancer and cancer stem cells. Asian Pac J Cancer Prev 2015; 16: 3629–3633.

    Article  Google Scholar 

  18. Lin ZS, Chu HC, Yen YC, Lewis BC, Chen YW . Kruppel-like factor 4, a tumor suppressor in hepatocellular carcinoma cells reverts epithelial mesenchymal transition by suppressing slug expression. PLoS One 2012; 7: e43593.

    Article  CAS  Google Scholar 

  19. Hu B, Wu Z, Liu T, Ullenbruch MR, Jin H, Phan SH . Gut-enriched Kruppel-like factor interaction with Smad3 inhibits myofibroblast differentiation. Am J Respir Cell Mol Biol 2007; 36: 78–84.

    Article  CAS  Google Scholar 

  20. Shields JM, Yang VW . Identification of the DNA sequence that interacts with the gut-enriched Kruppel-like factor. Nucleic Acids Res 1998; 26: 796–802.

    Article  CAS  Google Scholar 

  21. Fleming NI, Jorissen RN, Mouradov D, Christie M, Sakthianandeswaren A, Palmieri M et al. SMAD2, SMAD3 and SMAD4 mutations in colorectal cancer. Cancer Res 2013; 73: 725–735.

    Article  CAS  Google Scholar 

  22. Chen YW, Hsiao PJ, Weng CC, Kuo KK, Kuo TL, Wu DC et al. SMAD4 loss triggers the phenotypic changes of pancreatic ductal adenocarcinoma cells. BMC Cancer 2014; 14: 181.

    Article  Google Scholar 

  23. Matsuzaki K, Date M, Furukawa F, Tahashi Y, Matsushita M, Sakitani K et al. Autocrine stimulatory mechanism by transforming growth factor beta in human hepatocellular carcinoma. Cancer Res 2000; 60: 1394–1402.

    CAS  PubMed  Google Scholar 

  24. Mima K, Okabe H, Ishimoto T, Nakagawa S, Hayashi H, Kuroki H et al. CD44s regulates the TGF-beta-mediated mesenchymal phenotype and is associated with poor prognosis in patients with hepatocellular carcinoma. Cancer Res 2012; 72: 3414–3423.

    Article  CAS  Google Scholar 

  25. Morris SM, Baek JY, Koszarek A, Kanngurn S, Knoblaugh SE, Grady WM . Transforming growth factor-beta signaling promotes hepatocarcinogenesis induced by p53 loss. Hepatology 2012; 55: 121–131.

    Article  CAS  Google Scholar 

  26. Li Q, Gao Y, Jia Z, Mishra L, Guo K, Li Z et al. Dysregulated Kruppel-like factor 4 and vitamin D receptor signaling contribute to progression of hepatocellular carcinoma. Gastroenterology 2012; 143: e1–e2.

    Google Scholar 

  27. Wei D, Kanai M, Jia Z, Le X, Xie K . Kruppel-like factor 4 induces p27Kip1 expression in and suppresses the growth and metastasis of human pancreatic cancer cells. Cancer Res 2008; 68: 4631–4639.

    Article  CAS  Google Scholar 

  28. Yan X, Chen YG . Smad7: not only a regulator, but also a cross-talk mediator of TGF-beta signalling. Biochem J 2011; 434: 1–10.

    Article  CAS  Google Scholar 

  29. Stolfi C, Marafini I, De Simone V, Pallone F, Monteleone G . The dual role of Smad7 in the control of cancer growth and metastasis. Int J Mol Sci 2013; 14: 23774–23790.

    Article  Google Scholar 

  30. Park YN, Chae KJ, Oh BK, Choi J, Choi KS, Park C . Expression of Smad7 in hepatocellular carcinoma and dysplastic nodules: resistance mechanism to transforming growth factor-beta. Hepatogastroenterology 2004; 51: 396–400.

    CAS  PubMed  Google Scholar 

  31. Xia H, Ooi LL, Hui KM . MicroRNA-216a/217-induced epithelial-mesenchymal transition targets PTEN and SMAD7 to promote drug resistance and recurrence of liver cancer. Hepatology 2013; 58: 629–641.

    Article  CAS  Google Scholar 

  32. Wang J, Zhao J, Chu ES, Mok MT, Go MY, Man K et al. Inhibitory role of Smad7 in hepatocarcinogenesis in mice and in vitro. J Pathol 2013; 230: 441–452.

    Article  CAS  Google Scholar 

  33. von Gersdorff G, Susztak K, Rezvani F, Bitzer M, Liang D, Böttinger EP . Smad3 and Smad4 mediate transcriptional activation of the human Smad7 promoter by transforming growth factor beta. J Biol Chem 2000; 275: 11320–11326.

    Article  CAS  Google Scholar 

  34. Sun HC, Li M, Lu JL, Yan DW, Zhou CZ, Fan JW et al. Overexpression of Forkhead box M1 protein associates with aggressive tumor features and poor prognosis of hepatocellular carcinoma. Oncol Rep 2011; 25: 1533–1539.

    Google Scholar 

  35. Zhou A, Lin K, Zhang S, Chen Y, Zhang N, Xue J et al. Nuclear GSK3β promotes tumorigenesis by phosphorylating KDM1A and inducing its deubiquitylation by USP22. Nat Cell Biol 2016; 18: 954–966.

    Article  CAS  Google Scholar 

  36. Zhang N, Wei P, Gong A, Chiu WT, Lee HT, Colman H et al. FoxM1 promotes β-catenin nuclear localization and controls Wnt target-gene expression and glioma tumorigenesis. Cancer Cell 2011; 20: 427–442.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Don Norwood for editorial comments. The work is supported in part by grants R01-CA129956 and R01-CA148954 from the National Institutes of Health (K Xie), grant #81101623 from the National Natural Science Foundation of China and grant #81101623 and #81672846 from Science and Technology Commission of Shanghai Municipality (H Sun).

Author information

Authors and Affiliations

  1. Department of Gastroenterology, Hepatology and Nutrition, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    H Sun, Z Jia & K Xie

  2. Department of General Surgery, Shanghai General Hospital, Shanghai, People’s Republic of China

    H Sun, Z Peng, L Zhong & S Zhao

  3. Department of Pathology, Shanghai Jiaotong University Affiliated Shanghai General Hospital, Shanghai, People’s Republic of China

    H Tang

  4. Department of Oncology and Tumor Institute, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, People’s Republic of China

    D Xie & Y Gao

  5. Liver Transplantation Section, Center for Organ Transplantation, Fudan University, Shanghai, People’s Republic of China

    Z Ma

  6. Tumor Hospital, Southern Medical University, Guangzhou, Guangdong, People’s Republic of China

    L Zeng & R Luo

Authors
  1. H Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Z Peng
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. H Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. D Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Z Jia
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. L Zhong
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. S Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Z Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Y Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. L Zeng
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. R Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. K Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Z Peng, R Luo or K Xie.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies this paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sun, H., Peng, Z., Tang, H. et al. Loss of KLF4 and consequential downregulation of Smad7 exacerbate oncogenic TGF-β signaling in and promote progression of hepatocellular carcinoma. Oncogene 36, 2957–2968 (2017). https://doi.org/10.1038/onc.2016.447

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2016.447

This article is cited by

Search

Advanced search

Quick links