SMAD4 exerts a tumor-promoting role in hepatocellular carcinoma

Article metrics



Further understanding of the molecular biology and pathogenesis of hepatocellular carcinoma (HCC) is crucial for future therapeutic development. SMAD4, recognized as an important tumor suppressor, is a central mediator of transforming growth factor beta (TGFB) and bone morphogenetic protein (BMP) signaling. This study investigated the role of SMAD4 in HCC. Nuclear localization of SMAD4 was observed in a cohort of 140 HCC patients using tissue microarray. HCC cell lines were used for functional assay in vitro and in immune-deficient mice. Nuclear SMAD4 levels were significantly increased in patient HCC tumors as compared with adjacent tissues. Knockdown of SMAD4 significantly reduced the efficiency of colony formation and migratory capacity of HCC cells in vitro and was incompatible with HCC tumor initiation and growth in mice. Knockdown of SMAD4 partially conferred resistance to the anti-growth effects of BMP ligand in HCC cells. Importantly, simultaneous elevation of SMAD4 and phosphorylated SMAD2/3 is significantly associated with poor patient outcome after surgery. Although high levels of SMAD4 can also mediate an antitumor function by coupling with phosphorylated SMAD1/5/8, this signaling, however, is absent in majority of our HCC patients. In conclusion, this study revealed a highly non-canonical tumor-promoting function of SMAD4 in HCC. The drastic elevation of nuclear SMAD4 in sub-population of HCC tumors highlights its potential as an outcome predictor for patient stratification and a target for personalized therapeutic development.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9


  1. 1

    Wakefield LM, Hill CS . Beyond TGFbeta: roles of other TGFbeta superfamily members in cancer. Nat Rev Cancer 2013; 13: 328–341.

  2. 2

    Hardwick JC, Kodach LL, Offerhaus GJ, van den Brink GR . Bone morphogenetic protein signalling in colorectal cancer. Nat Rev Cancer 2008; 8: 806–812.

  3. 3

    Shi Y, Massague J . Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 2003; 113: 685–700.

  4. 4

    Shi Y, Hata A, Lo RS, Massague J, Pavletich NP . A structural basis for mutational inactivation of the tumour suppressor Smad4. Nature 1997; 388: 87–93.

  5. 5

    Lagna G, Hata A, Hemmati-Brivanlou A, Massague J . Partnership between DPC4 and SMAD proteins in TGF-beta signalling pathways. Nature 1996; 383: 832–836.

  6. 6

    Hahn SA, Hoque AT, Moskaluk CA, da Costa LT, Schutte M, Rozenblum E et al. Homozygous deletion map at 18q21.1 in pancreatic cancer. Cancer Res 1996; 56: 490–494.

  7. 7

    Xu X, Kobayashi S, Qiao W, Li C, Xiao C, Radaeva S et al. Induction of intrahepatic cholangiocellular carcinoma by liver-specific disruption of Smad4 and Pten in mice. J Clin Invest 2006; 116: 1843–1852.

  8. 8

    Ding Z, Wu CJ, Chu GC, Xiao Y, Ho D, Zhang J et al. SMAD4-dependent barrier constrains prostate cancer growth and metastatic progression. Nature 2011; 470: 269–273.

  9. 9

    Zhang B, Halder SK, Kashikar ND, Cho YJ, Datta A, Gorden DL et al. Antimetastatic role of Smad4 signaling in colorectal cancer. Gastroenterology 2010; 138: 969–980 e961-963.

  10. 10

    Itatani Y, Kawada K, Fujishita T, Kakizaki F, Hirai H, Matsumoto T et al. Loss of SMAD4 from colorectal cancer cells promotes CCL15 expression to recruit CCR1+ myeloid cells and facilitate liver metastasis. Gastroenterology 2013; 145: 1064–1075 e1011.

  11. 11

    Argani P, Shaukat A, Kaushal M, Wilentz RE, Su GH, Sohn TA et al. Differing rates of loss of DPC4 expression and of p53 overexpression among carcinomas of the proximal and distal bile ducts. Cancer 2001; 91: 1332–1341.

  12. 12

    Caputo V, Cianetti L, Niceta M, Carta C, Ciolfi A, Bocchinfuso G et al. A restricted spectrum of mutations in the SMAD4 tumor-suppressor gene underlies Myhre syndrome. Am J Hum Genet 2012; 90: 161–169.

  13. 13

    Korsse SE, Biermann K, Offerhaus GJ, Wagner A, Dekker E, Mathus-Vliegen EM et al. Identification of molecular alterations in gastrointestinal carcinomas and dysplastic hamartomas in Peutz-Jeghers syndrome. Carcinogenesis 2013; 34: 1611–1619.

  14. 14

    Tangkijvanich P, Anukulkarnkusol N, Suwangool P, Lertmaharit S, Hanvivatvong O, Kullavanijaya P et al. Clinical characteristics and prognosis of hepatocellular carcinoma: analysis based on serum alpha-fetoprotein levels. J Clin Gastroenterol 2000; 31: 302–308.

  15. 15

    Kocabayoglu P, Friedman SL . Cellular basis of hepatic fibrosis and its role in inflammation and cancer. Front Biosci (Schol Ed) 2013; 5: 217–230.

  16. 16

    Ikushima H, Miyazono K . TGFbeta signalling: a complex web in cancer progression. Nat Rev Cancer 2010; 10: 415–424.

  17. 17

    Massague J . TGFbeta signalling in context. Nat Rev Mol Cell Biol 2012; 13: 616–630.

  18. 18

    Matsuzaki K . Smad phospho-isoforms direct context-dependent TGF-beta signaling. Cytokine Growth Factor Rev 2013; 24: 385–399.

  19. 19

    Furukawa F, Matsuzaki K, Mori S, Tahashi Y, Yoshida K, Sugano Y et al. p38 MAPK mediates fibrogenic signal through Smad3 phosphorylation in rat myofibroblasts. Hepatology 2003; 38: 879–889.

  20. 20

    Sekimoto G, Matsuzaki K, Yoshida K, Mori S, Murata M, Seki T et al. Reversible Smad-dependent signaling between tumor suppression and oncogenesis. Cancer Res 2007; 67: 5090–5096.

  21. 21

    Mori S, Matsuzaki K, Yoshida K, Furukawa F, Tahashi Y, Yamagata H et al. TGF-beta and HGF transmit the signals through JNK-dependent Smad2/3 phosphorylation at the linker regions. Oncogene 2004; 23: 7416–7429.

  22. 22

    Matsuzaki K, Murata M, Yoshida K, Sekimoto G, Uemura Y, Sakaida N et al. Chronic inflammation associated with hepatitis C virus infection perturbs hepatic transforming growth factor beta signaling, promoting cirrhosis and hepatocellular carcinoma. Hepatology 2007; 46: 48–57.

  23. 23

    Murata M, Matsuzaki K, Yoshida K, Sekimoto G, Tahashi Y, Mori S et al. Hepatitis B virus X protein shifts human hepatic transforming growth factor (TGF)-beta signaling from tumor suppression to oncogenesis in early chronic hepatitis B. Hepatology 2009; 49: 1203–1217.

  24. 24

    Zhang L, Sun H, Zhao F, Lu P, Ge C, Li H et al. BMP4 administration induces differentiation of CD133+ hepatic cancer stem cells, blocking their contributions to hepatocellular carcinoma. Cancer Res 2012; 72: 4276–4285.

  25. 25

    Piccirillo SG, Reynolds BA, Zanetti N, Lamorte G, Binda E, Broggi G et al. Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells. Nature 2006; 444: 761–765.

  26. 26

    Wang L, Park P, Zhang H, La Marca F, Claeson A, Than K et al. BMP-2 inhibits tumor growth of human renal cell carcinoma and induces bone formation. Int J Cancer 2012; 131: 1941–1950.

  27. 27

    Virtanen S, Alarmo EL, Sandstrom S, Ampuja M, Kallioniemi A . Bone morphogenetic protein -4 and -5 in pancreatic cancer—novel bidirectional players. Exp Cell Res 2011; 317: 2136–2146.

  28. 28

    Hahn SA, Seymour AB, Hoque AT, Schutte M, da Costa LT, Redston MS et al. Allelotype of pancreatic adenocarcinoma using xenograft enrichment. Cancer Res 1995; 55: 4670–4675.

  29. 29

    Hahn SA, Schutte M, Hoque AT, Moskaluk CA, da Costa LT, Rozenblum E et al. DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science 1996; 271: 350–353.

  30. 30

    Thiagalingam S, Lengauer C, Leach FS, Schutte M, Hahn SA, Overhauser J et al. Evaluation of candidate tumour suppressor genes on chromosome 18 in colorectal cancers. Nat Genet 1996; 13: 343–346.

  31. 31

    Kang YK, Kim WH, Jang JJ . Expression of G1-S modulators (p53, p16, p27, cyclin D1, Rb) and Smad4/Dpc4 in intrahepatic cholangiocarcinoma. Hum Pathol 2002; 33: 877–883.

  32. 32

    Yao L, Li FJ, Tang ZQ, Gao S, Wu QQ . Smad4 expression in hepatocellular carcinoma differs by hepatitis status. Asian Pac J Cancer Prev 2012; 13: 1297–1303.

  33. 33

    Hiwatashi K, Ueno S, Sakoda M, Kubo F, Tateno T, Kurahara H et al. Strong Smad4 expression correlates with poor prognosis after surgery in patients with hepatocellular carcinoma. Ann Surg Oncol 2009; 16: 3176–3182.

  34. 34

    Kodach LL, Bleuming SA, Peppelenbosch MP, Hommes DW, van den Brink GR, Hardwick JC . The effect of statins in colorectal cancer is mediated through the bone morphogenetic protein pathway. Gastroenterology 2007; 133: 1272–1281.

  35. 35

    Kodach LL, Bleuming SA, Musler AR, Peppelenbosch MP, Hommes DW, van den Brink GR et al. The bone morphogenetic protein pathway is active in human colon adenomas and inactivated in colorectal cancer. Cancer 2008; 112: 300–306.

  36. 36

    Guo X, Wang XF . Signaling cross-talk between TGF-beta/BMP and other pathways. Cell Res 2009; 19: 71–88.

  37. 37

    Piek E, Roberts AB . Suppressor and oncogenic roles of transforming growth factor-beta and its signaling pathways in tumorigenesis. Adv Cancer Res 2001; 83: 1–54.

  38. 38

    Mullauer L, Grasl-Kraupp B, Bursch W, Schulte-Hermann R . Transforming growth factor beta 1-induced cell death in preneoplastic foci of rat liver and sensitization by the antiestrogen tamoxifen. Hepatology 1996; 23: 840–847.

  39. 39

    Abou-Shady M, Baer HU, Friess H, Berberat P, Zimmermann A, Graber H et al. Transforming growth factor betas and their signaling receptors in human hepatocellular carcinoma. Am J Surg 1999; 177: 209–215.

  40. 40

    Matsuzaki K, Kitano C, Murata M, Sekimoto G, Yoshida K, Uemura Y et al. Smad2 and Smad3 phosphorylated at both linker and COOH-terminal regions transmit malignant TGF-beta signal in later stages of human colorectal cancer. Cancer Res 2009; 69: 5321–5330.

  41. 41

    Thawani JP, Wang AC, Than KD, Lin CY, La Marca F, Park P . Bone morphogenetic proteins and cancer: review of the literature. Neurosurgery 2010; 66: 233–246 discussion 246.

  42. 42

    Maegdefrau U, Bosserhoff AK . BMP activated Smad signaling strongly promotes migration and invasion of hepatocellular carcinoma cells. Exp Mol Pathol 2012; 92: 74–81.

  43. 43

    Chiu CY, Kuo KK, Kuo TL, Lee KT, Cheng KH . The activation of MEK/ERK signaling pathway by bone morphogenetic protein 4 to increase hepatocellular carcinoma cell proliferation and migration. Mol Cancer Res 2012; 10: 415–427.

  44. 44

    Carreira AC, Lojudice FH, Halcsik E, Navarro RD, Sogayar MC, Granjeiro JM . Bone morphogenetic proteins: facts, challenges, and future perspectives. J Dent Res 2014; 93: 335–345.

  45. 45

    Sakai H, Furihata M, Matsuda C, Takahashi M, Miyazaki H, Konakahara T et al. Augmented autocrine bone morphogenic protein (BMP) 7 signaling increases the metastatic potential of mouse breast cancer cells. Clin Exp Metastasis 2012; 29: 327–338.

  46. 46

    Li Q, Gu X, Weng H, Ghafoory S, Liu Y, Feng T et al. Bone morphogenetic protein-9 induces epithelial to mesenchymal transition in hepatocellular carcinoma cells. Cancer Sci 2013; 104: 398–408.

  47. 47

    Pedroza-Gonzalez A, Verhoef C, Ijzermans JN, Peppelenbosch MP, Kwekkeboom J, Verheij J et al. Activated tumor-infiltrating CD4+ regulatory T cells restrain antitumor immunity in patients with primary or metastatic liver cancer. Hepatology 2013; 57: 183–194.

  48. 48

    Hernanda PY, Pedroza-Gonzalez A, van der Laan LJ, Broker ME, Hoogduijn MJ, Ijzermans JN et al. Tumor promotion through the mesenchymal stem cell compartment in human hepatocellular carcinoma. Carcinogenesis 2013; 34: 2330–2340.

  49. 49

    Pan Q, de Ruiter PE, von Eije KJ, Smits R, Kwekkeboom J, Tilanus HW et al. Disturbance of the microRNA pathway by commonly used lentiviral shRNA libraries limits the application for screening host factors involved in hepatitis C virus infection. FEBS Lett 2011; 585: 1025–1030.

  50. 50

    van Horssen R, Galjart N, Rens JA, Eggermont AM, ten Hagen TL . Differential effects of matrix and growth factors on endothelial and fibroblast motility: application of a modified cell migration assay. J Cell Biochem 2006; 99: 1536–1552.

  51. 51

    Das AM, Seynhaeve AL, Rens JA, Vermeulen CE, Koning GA, Eggermont AM et al. Differential TIMP3 expression affects tumor progression and angiogenesis in melanomas through regulation of directionally persistent endothelial cell migration. Angiogenesis 2014; 17: 163–177.

  52. 52

    Pan Q, Liu B, Liu J, Cai R, Liu X, Qian C . Synergistic antitumor activity of XIAP-shRNA and TRAIL expressed by oncolytic adenoviruses in experimental HCC. Acta Oncol 2008; 47: 135–144.

Download references


We thank the support from the Daniel den Hoed Foundation for a Centennial Award fellowship (to Q Pan), the Netherlands Organization for Scientific Research (NWO/ZonMw) for a VENI grant (no. 916-13-032) (to Q Pan), the Dutch Digestive Foundation (MLDS) for a career development grant (no. CDG 1304) (to Q Pan) and the European Association for the Study of the Liver (EASL) for a Sheila Sherlock Fellowship (to Q Pan). Support from the Science and Technology Department Commonwealth Technology Applied Research Project (no. 2012F82G2060018) of Zhejiang Province, China and the National Nature Science Foundation of China (No. 81272687) (to K Chen) is gratefully acknowledged. We thank Dr Ron Smits from Erasmus Medical Center Rotterdam for critical reading of the manuscript and thank Dr Jie Xu from the Animal Care at Hangzhou Normal University, Hangzhou, China for helping with the animal experiments.

Author information

Correspondence to Q Pan.

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

Verify currency and authenticity via CrossMark

Further reading