Skip to main content

Advertisement

SpringerLink
Log in

miR-675 Mediates Downregulation of Twist1 and Rb in AFP-Secreting Hepatocellular Carcinoma

  • Translational Research and Biomarkers
  • Published:
Annals of Surgical Oncology Aims and scope Submit manuscript

Abstract

Background

Alpha-fetoprotein (AFP)-secreting hepatocellular carcinomas (HCC) represent a genetically distinct subset of tumors often associated with a worse prognosis. However, the molecular mechanisms that underlie these phenotypic differences remain poorly understood.

Methods

HCC tumor samples from 27 patients were profiled using the Affymetrix 133 Plus 2.0 GeneChips. GeneGO Metacore software was used to identify altered biologic pathways. Expression validation was confirmed by RT-PCR. Manipulation of miR-675 by overexpression and antagomir-mediated knockdown was carried out with subsequent evaluation of effects on cell behavior by cell cycle, proliferation, invasion, and growth in soft agar assays.

Results

We identified a strong relationship between primary tumor H19 gene expression and elevated serum AFP. H19 has recently been identified to encode microRNA-675 (miR-675), and we confirmed the relationship in an independent sample of patients. Pathway analyses of the effect of miR-675 overexpression in hepatoma cells revealed a predominant upregulation of cell adhesion and cell cycle initiation pathways. We have demonstrated that miR-675 mediates increases in proliferation and an accumulation of cells with tetraploid DNA content associated with a repression of Rb. We also demonstrated that overexpression of miR-675 alters cellular morphology, reduces invasive potential, and increases anchorage-independent growth capacity. These findings are consistent with a mesenchymal-to-epithelial transition, associated with a reduction in the expression of the key EMT mediator, Twist1.

Conclusions

Expression of the miR-675 in hepatocellular carcinoma links a dramatic upregulation of proliferative and growth capacity with inhibition of motility in HCC cells.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Ferlay J, Shin HR, Bray F, Forman D, Mathers C, et al. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 2010;127:2893–7.

    Article  PubMed  CAS  Google Scholar 

  2. Benson AB, 3rd, Abrams TA, Ben-Josef E, Bloomston PM, Botha JF, et al. NCCN clinical practice guidelines in oncology: hepatobiliary cancers. J Natl Compr Cancer Netw. 2009;7:350–91.

    CAS  Google Scholar 

  3. Tangkijvanich P, Anukulkarnkusol N, Suwangool P, Lertmaharit S, Hanvivatvong O, et al. Clinical characteristics and prognosis of hepatocellular carcinoma: analysis based on serum alpha-fetoprotein levels. J Clin Gastroenterol. 2000;31:302–8.

    Article  PubMed  CAS  Google Scholar 

  4. Hoshida Y, Nijman SM, Kobayashi M, Chan JA, Brunet JP, et al. Integrative transcriptome analysis reveals common molecular subclasses of human hepatocellular carcinoma. Cancer Res. 2009;69:7385–92.

    Article  PubMed  CAS  Google Scholar 

  5. Takayasu K, Arii S, Ikai I, Omata M, Okita K, et al. Prospective cohort study of transarterial chemoembolization for unresectable hepatocellular carcinoma in 8510 patients. Gastroenterology 2006;131:461–9.

    Article  PubMed  CAS  Google Scholar 

  6. Taketa K Alpha-fetoprotein: reevaluation in hepatology. Hepatology 1990;12:1420–32.

    Article  PubMed  CAS  Google Scholar 

  7. Budhu A, Forgues M, Ye QH, Jia HL, He P, et al. Prediction of venous metastases, recurrence, and prognosis in hepatocellular carcinoma based on a unique immune response signature of the liver microenvironment. Cancer Cell 2006;10:99–111.

    Article  PubMed  CAS  Google Scholar 

  8. Lee JS, Heo J, Libbrecht L, Chu IS, Kaposi-Novak P, et al. A novel prognostic subtype of human hepatocellular carcinoma derived from hepatic progenitor cells. Nat Med. 2006;12:410–6.

    Article  PubMed  CAS  Google Scholar 

  9. Wurmbach E, Chen YB, Khitrov G, Zhang W, Roayaie S, et al. Genome-wide molecular profiles of HCV-induced dysplasia and hepatocellular carcinoma. Hepatology 2007;45:938–47.

    Article  PubMed  CAS  Google Scholar 

  10. Dudich E, Semenkova L, Gorbatova E, Dudich I, Khromykh L, et al. Growth-regulative activity of human alpha-fetoprotein for different types of tumor and normal cells. Tumour Biol. 1998;19:30–40.

    Article  PubMed  CAS  Google Scholar 

  11. Li MS, Li PF, Yang FY, He SP, Du GG, et al. The intracellular mechanism of alpha-fetoprotein promoting the proliferation of NIH 3T3 cells. Cell Res. 2002;12:151–6.

    Article  PubMed  Google Scholar 

  12. Wang XW, Xu B Stimulation of tumor-cell growth by alpha-fetoprotein. Int J Cancer 1998;75:596–9.

    Article  PubMed  CAS  Google Scholar 

  13. Fuchs BC, Fujii T, Dorfman JD, Goodwin JM, Zhu AX, et al. Epithelial-to-mesenchymal transition and integrin-linked kinase mediate sensitivity to epidermal growth factor receptor inhibition in human hepatoma cells. Cancer Res. 2008;68:2391–9.

    Article  PubMed  CAS  Google Scholar 

  14. Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, et al. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 2003;4:249–64.

    Article  PubMed  Google Scholar 

  15. Elahi A, Zhang L, Yeatman TJ, Gery S, Sebti S, et al. HPP1-mediated tumor suppression requires activation of STAT1 pathways. Int J Cancer 2008;122:1567–72.

    Article  PubMed  CAS  Google Scholar 

  16. Tsang WP, Ng EK, Ng SS, Jin H, Yu J, et al. Oncofetal H19-derived miR-675 regulates tumor suppressor RB in human colorectal cancer. Carcinogenesis 2010;31:350–8.

    Article  PubMed  CAS  Google Scholar 

  17. Yoshimizu T, Miroglio A, Ripoche MA, Gabory A, Vernucci M, et al. The H19 locus acts in vivo as a tumor suppressor. Proc Natl Acad Sci USA 2008;105:12417–22.

    Article  PubMed  CAS  Google Scholar 

  18. Berteaux N, Lottin S, Monte D, Pinte S, Quatannens B, et al. H19 mRNA-like noncoding RNA promotes breast cancer cell proliferation through positive control by E2F1. J Biol Chem. 2005;280:29625–36.

    Article  PubMed  CAS  Google Scholar 

  19. Matouk IJ, Mezan S, Mizrahi A, Ohana P, Abu-Lail R, et al. The oncofetal H19 RNA connection: hypoxia, p53 and cancer. Biochim Biophys Acta 2010;1803:443–51.

    Article  PubMed  CAS  Google Scholar 

  20. Gentric G, Desdouets C, Celton-Morizur S Hepatocytes polyploidization and cell cycle control in liver physiopathology. Int J Hepatol. 2012;28:2430.

    Google Scholar 

  21. Margolis RL Tetraploidy and tumor development. Cancer Cell 2005;8:353–4.

    Article  PubMed  CAS  Google Scholar 

  22. Gabory A, Jammes H, Dandolo L The H19 locus: role of an imprinted non-coding RNA in growth and development. Bioessays 2010;32:473–80.

    Article  PubMed  CAS  Google Scholar 

  23. Wu J, Qin Y, Li B, He WZ, Sun ZL Hypomethylated and hypermethylated profiles of H19DMR are associated with the aberrant imprinting of IGF2 and H19 in human hepatocellular carcinoma. Genomics 2008;91:443–50.

    Article  PubMed  CAS  Google Scholar 

  24. Li J, Ran C, Li E, Gordon F, Comstock G, et al. Synergistic function of E2F7 and E2F8 is essential for cell survival and embryonic development. Dev Cell 2008;14:62–75.

    Article  PubMed  CAS  Google Scholar 

  25. Lottin S, Adriaenssens E, Dupressoir T, Berteaux N, Montpellier C, et al. Overexpression of an ectopic H19 gene enhances the tumorigenic properties of breast cancer cells. Carcinogenesis 2002;23:1885–95.

    Article  PubMed  CAS  Google Scholar 

  26. Kruger J, Rehmsmeier M RNAhybrid: microRNA target prediction easy, fast and flexible. Nucl Acids Res. 2006;34:W451–4.

    Article  PubMed  Google Scholar 

  27. Baum B, Settleman J, Quinlan MP Transitions between epithelial and mesenchymal states in development and disease. Semin Cell Dev Biol. 2008;19:294–308.

    Article  PubMed  CAS  Google Scholar 

  28. Thiery JP, Sleeman JP Complex networks orchestrate epithelial–mesenchymal transitions. Nat Rev Mol Cell Biol. 2006;7:131–42.

    Article  PubMed  CAS  Google Scholar 

  29. Lee TK, Poon RT, Yuen AP, Ling MT, Kwok WK, et al. Twist overexpression correlates with hepatocellular carcinoma metastasis through induction of epithelial–mesenchymal transition. Clin Cancer Res. 2006;12:5369–76.

    Article  PubMed  CAS  Google Scholar 

  30. Matsuo N, Shiraha H, Fujikawa T, Takaoka N, Ueda N, et al. Twist expression promotes migration and invasion in hepatocellular carcinoma. BMC Cancer 2009;9:240.

    Article  PubMed  Google Scholar 

  31. Gunasinghe NP, Wells A, Thompson EW, Hugo HJ Mesenchymal–epithelial transition (MET) as a mechanism for metastatic colonisation in breast cancer. Cancer Metastasis Rev. 2012; 31:469–78.

    Google Scholar 

  32. Lustig-Yariv O, Schulze E, Komitowski D, Erdmann V, Schneider T, et al. The expression of the imprinted genes H19 and IGF-2 in choriocarcinoma cell lines. Is H19 a tumor suppressor gene? Oncogene 1997;15:169–77.

    Article  PubMed  CAS  Google Scholar 

  33. Rachmilewitz J, Elkin M, Rosensaft J, Gelman-Kohan Z, Ariel I, et al. H19 expression and tumorigenicity of choriocarcinoma derived cell lines. Oncogene 1995;11:863–70.

    PubMed  CAS  Google Scholar 

  34. Yang J, Mani SA, Donaher JL, Ramaswamy S, Itzykson RA, et al. Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell 2004;117:927–39.

    Article  PubMed  CAS  Google Scholar 

  35. Yang MH, Chen CL, Chau GY, Chiou SH, Su CW, et al. Comprehensive analysis of the independent effect of twist and snail in promoting metastasis of hepatocellular carcinoma. Hepatology 2009;50:1464–74.

    Article  PubMed  CAS  Google Scholar 

  36. Hugo H, Ackland ML, Blick T, Lawrence MG, Clements JA, et al. Epithelial–mesenchymal and mesenchymal–epithelial transitions in carcinoma progression. J Cell Physiol. 2007;213:374–83.

    Article  PubMed  CAS  Google Scholar 

  37. Yang J, Weinberg RA Epithelial–mesenchymal transition: at the crossroads of development and tumor metastasis. Dev Cell 2008;14:818–29.

    Article  PubMed  CAS  Google Scholar 

  38. Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, et al. The epithelial–mesenchymal transition generates cells with properties of stem cells. Cell 2008;133:704–15.

    Article  PubMed  CAS  Google Scholar 

  39. Morel AP, Lievre M, Thomas C, Hinkal G, Ansieau S, et al. Generation of breast cancer stem cells through epithelial–mesenchymal transition. PLoS One 2008;3:e2888.

    Article  PubMed  Google Scholar 

  40. Peinado H, Olmeda D, Cano A Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? Nat Rev Cancer 2007;7:415–28.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgment

This work was supported in part by a Grant from the National Institutes of Health (NIH) (R01 CA112215 to J.M.H. and T.Y.)

Author information

Authors and Affiliations

  1. Department of Gastrointestinal Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA

    J. M. Hernandez, A. Elahi, C. W. Clark, J. Wang, L. A. Humphries, T. Yeatman & D. Shibata

  2. Department of Anatomic Pathology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA

    B. Centeno

  3. Department of Biomedical Informatics, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA

    G. Bloom

  4. Division of Surgical Oncology, Massachusetts General Hospital Cancer Center, Boston, MA, USA

    B. C. Fuchs

  5. Division of Surgical Oncology, Harvard Medical School, Boston, MA, USA

    B. C. Fuchs

Authors
  1. J. M. Hernandez
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. A. Elahi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. C. W. Clark
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. J. Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. L. A. Humphries
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. B. Centeno
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. G. Bloom
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. B. C. Fuchs
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. T. Yeatman
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. D. Shibata
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to D. Shibata.

Additional information

J. M. Hernandez and A. Elahi contributed equally to this work and therefore share first authorship.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 24 kb)

10434_2013_3106_MOESM2_ESM.jpg

Supplemental Fig. 1 Kaplan–Meier survival curves for AFP-secreting and non-AFP-secreting HCC. Note: four patients were lost to follow-up and therefore were not included in the analysis. (JPEG 1238 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hernandez, J.M., Elahi, A., Clark, C.W. et al. miR-675 Mediates Downregulation of Twist1 and Rb in AFP-Secreting Hepatocellular Carcinoma. Ann Surg Oncol 20 (Suppl 3), 625–635 (2013). https://doi.org/10.1245/s10434-013-3106-3

Download citation

  • Received:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1245/s10434-013-3106-3

Keywords

Search

Navigation