Tumor Biology

, Volume 37, Issue 1, pp 911–924 | Cite as

In vivo and in vitro effect of hepatocarcinoma lymph node metastasis by upregulation of Annexin A7 and relevant mechanisms

  • Xian-Yan Wang
  • Feng Gao
  • Yu-Rong Sun
  • Lu-Lu Bai
  • Mohammed Mohammed Ibrahim
  • Bo Wang
  • Jian-Wu Tang
Research Article


We unveiled the association of Annexin A7 with vascular endothelial growth factor-C (VEGF-C) and the effect of upregulation of Annexin A7 in Hca-F and Hca-P cells on inhibiting hepatocarcinoma (HCC) lymph node metastasis (LNM) in vitro and in vivo. A total of 200 inbred 615 mice were randomly divided into four equal groups inoculated with Hca-F, Hca-P, FAnxa7-upregulated, and PAnxa7-upregulated cells, respectively. The primary tumor, popliteal, inguinal, and iliac lymph nodes were prepared for immunohistochemical (IHC) staining, real-time quantitative polymerase chain reaction (qRT-PCR) analysis, Western blot, and hematoxylin-eosin (H&E) staining. There was over 50 % increase both in the number of FAnxa7-upregulated and PAnxa7-upregulated cells migrated through the filter compared to their controls (FAnxa7-control, Hca-F and PAnxa7-control, Hca-P). However, no significant differences were noted in invasion ability between them (all P > 0.05). Tumor lymph vessels were significantly reduced in FAnxa7-upregulated and PAnxa7-upregulated tumors when compared with Hca-F and Hca-P tumors (all P < 0.05). Blood vessel density did not differ significantly between FAnxa7-upregulated and PAnxa7-upregulated tumors and Hca-F and Hca-P tumors. Enzyme-linked immunosorbent assay (ELISA) for VEGF-C showed that upregulating Annexin A7 decreased VEGF-C secretion in FAnxa7-upregulated and PAnxa7-upregulated cells (P < 0.05). The IHC staining result showed that the level of serum Annexin A7 was found to be statistically higher in all experimental groups than that in the control group (P < 0.05). The present results indicated that alterations in serum Annexin A7 expression may be of prognostic relevance in HCC lymphatic metastasis.


Hepatocarcinoma Annexin A7 Vascular endothelial growth factor-C Lymph node metastasis 



This work was supported by the National Natural Science Foundation of China (nos. 30772468 and 81071725) and the Educational Department of Liaoning Province (nos. 2008225010-3, 2007-T024, and 2009S028). This study was also supported by the Key Laboratory of Tumor Metastasis of Liaoning Province.

Conflicts of interest



  1. 1.
    Jiang DK, Sun J, Cao G, Liu Y, Lin D, Gao YZ, et al. Genetic variants in STAT4 and HLA-DQ genes confer risk of hepatitis B virus-related hepatocellular carcinoma. Nat Genet. 2013;45:72–5.CrossRefPubMedGoogle Scholar
  2. 2.
    Fujimoto A, Totoki Y, Abe T, Boroevich KA, Hosoda F, Nguyen HH, et al. Whole-genome sequencing of liver cancers identifies etiological influences on mutation patterns and recurrent mutations in chromatin regulators. Nat Genet. 2012;44:760–4.CrossRefPubMedGoogle Scholar
  3. 3.
    El-Serag HB. Epidemiology of viral hepatitis and hepatocellular carcinoma. Gastroenterology. 2012;142:1264–73.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Wakamatsu Y, Sakamoto N, Oo HZ, Naito Y, Uraoka N, Anami K, et al. Expression of cancer stem cell markers ALDH1, CD44 and CD133 in primary tumor and lymph node metastasis of gastric cancer. Pathol Int. 2012;62:112–9.CrossRefPubMedGoogle Scholar
  5. 5.
    Patel V, Marsh CA, Dorsam RT, Mikelis CM, Masedunskas A, Amornphimoltham P, et al. Decreased lymphangiogenesis and lymph node metastasis by mTOR inhibition in head and neck cancer. Cancer Res. 2011;71:7103–12.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Shen XH, Li HK, Wang F, Zhang T, Cui YL, Li Q. Clinical significance of lymph node metastasis in patients undergoing partial hepatectomy for hepatocellular carcinoma. World J Surg. 2010;34:1028–33.CrossRefGoogle Scholar
  7. 7.
    Xiang ZL, Zeng ZC, Fan J, Tang ZY, Zeng HY, Gao DM. Gene expression profiling of fixed tissues identified hypoxia-inducible factor-1alpha, VEGF, and matrix metalloproteinase-2 as biomarkers of lymph node metastasis in hepatocellular carcinoma. Clin Cancer Res. 2011;17:5463–72.CrossRefPubMedGoogle Scholar
  8. 8.
    Gebo KA, Chander G, Jenckes MW, Ghanem KG, Herlong HF, Torbenson MS, et al. Screening tests for hepatocellular carcinoma in patients with chronic hepatitis C: a systematic review. Hepatology. 2002;36:S84–92.CrossRefPubMedGoogle Scholar
  9. 9.
    Guo C, Liu S, Greenaway F, Sun MZ. Potential role of annexin A7 in cancers. Clin Chim Acta. 2013;423:83–9.CrossRefPubMedGoogle Scholar
  10. 10.
    Liu S, Sun MZ, Tang JW, Wang Z, Sun C, Greenaway FT. High-performance liquid chromatography/nano-electrospray ionization tandem mass spectrometry, two-dimensional difference in-gel electrophoresis and gene microarray identification of lymphatic metastasis-associated biomarkers. Rapid Commun Mass Spectrom. 2008;22:3172–8.CrossRefPubMedGoogle Scholar
  11. 11.
    Song B, Tang JW, Wang B, Cui XN, Hou L, Sun L, et al. Identify lymphatic metastasis-associated genes in mouse hepatocarcinoma cell lines using gene chip. World J Gastroenterol. 2005;11:1463–72.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Jin YL, Wang ZQ, Qu H, Wang HX, Ibrahim MM, Zhang J, et al. Annexin A7 gene is an important factor in the lymphatic metastasis of tumors. Biomed Pharmacother. 2013;67:251–9.CrossRefPubMedGoogle Scholar
  13. 13.
    Srivastava M, Leighton X, Starr J, Eidelman O, Pollard HB. Diverse effects of ANXA7 and p53 on LNCaP prostate cancer cells are associated with regulation of SGK1 transcription and phosphorylation of the SGK1 target FOXO3A. Biomed Res Int. 2014;2014:193635.PubMedPubMedCentralGoogle Scholar
  14. 14.
    Huang Y, Wang Q, Du Y, Bai L, Jin F, Zhang J, et al. Inhibition of annexin A7 gene and protein induces the apotosis and decreases the invasion, migration of the hepatocarcinoma cell line. Biomed Pharmacother. 2014;68:819–24.CrossRefPubMedGoogle Scholar
  15. 15.
    Villefranc JA, Nicoli S, Bentley K, Jeltsch M, Zarkada G, Moore JC, et al. A truncation allele in vascular endothelial growth factor c reveals distinct modes of signaling during lymphatic and vascular development. Development. 2013;140:1497–506.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Tammela T, Saaristo A, Holopainen T, Lyytikka J, Kotronen A, Pitkonen M, et al. Therapeutic differentiation and maturation of lymphatic vessels after lymph node dissection and transplantation. Nat Med. 2007;13:1458–66.CrossRefPubMedGoogle Scholar
  17. 17.
    Ozasa R, Ohno J, Iwahashi T, Taniguchi K. Tumor-induced lymphangiogenesis in cervical lymph nodes in oral melanoma-bearing mice. J Exp Clin Cancer Res. 2012;31:83.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Cheng D, Liang B, Li Y. Serum vascular endothelial growth factor (VEGF-C) as a diagnostic and prognostic marker in patients with ovarian cancer. PLoS One. 2013;8, e55309.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Wang CA, Jedlicka P, Patrick AN, Micalizzi DS, Lemmer KC, Deitsch E, et al. SIX1 induces lymphangiogenesis and metastasis via upregulation of VEGF-C in mouse models of breast cancer. J Clin Invest. 2012;122:1895–906.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Gou HF, Chen XC, Zhu J, Jiang M, Yang Y, Cao D, et al. Expressions of COX-2 and VEGF-C in gastric cancer: correlations with lymphangiogenesis and prognostic implications. J Exp Clin Cancer Res. 2011;30:14.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Xiang Z, Zeng Z, Tang Z, Fan J, Sun H, Wu W, et al. Increased expression of vascular endothelial growth factor-C and nuclear CXCR4 in hepatocellular carcinoma is correlated with lymph node metastasis and poor outcome. Cancer J. 2009;15:519–25.CrossRefPubMedGoogle Scholar
  22. 22.
    Acs G, Paragh G, Rakosy Z, Laronga C, Zhang PJ. The extent of retraction clefts correlates with lymphatic vessel density and VEGF-C expression and predicts nodal metastasis and poor prognosis in early-stage breast carcinoma. Mod Pathol. 2012;25:163–77.CrossRefPubMedGoogle Scholar
  23. 23.
    Min Y, Ghose S, Boelte K, Li J, Yang L, Lin PC. C/EBP-delta regulates VEGF-C autocrine signaling in lymphangiogenesis and metastasis of lung cancer through HIF-1alpha. Oncogene. 2011;30:4901–9.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Khromova N, Kopnin P, Rybko V, Kopnin BP. Downregulation of VEGF-C expression in lung and colon cancer cells decelerates tumor growth and inhibits metastasis via multiple mechanisms. Oncogene. 2012;31:1389–97.CrossRefPubMedGoogle Scholar
  25. 25.
    Chen H, Zhao GP, Xiao NX, Xia Q, Lai JS, Zheng DS, et al. Correlation of the expressions of VEGF-C and VEGFR-3 to the pathological grade of prostate cancer. Nan Fang Yi Ke Da Xue Xue Bao. 2011;31:155–9.PubMedGoogle Scholar
  26. 26.
    Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods. 2001;25:402–8.CrossRefPubMedGoogle Scholar
  27. 27.
    Feldman JP, Goldwasser R, Mark S, Schwartz J, Orion I. A mathematical model for tumor volume evaluation using two-dimensions. J Appl Quant Methods. 2009;4:455–62.Google Scholar
  28. 28.
    Zhang J, Song M, Wang J, Sun M, Wang B, Li R, et al. Enoyl coenzyme A hydratase 1 is an important factor in the lymphatic metastasis of tumors. Biomed Pharmacother. 2011;65:157–62.CrossRefPubMedGoogle Scholar
  29. 29.
    Jin Y, Mao J, Wang H, Hou Z, Ma W, Zhang J, et al. Enhanced tumorigenesis and lymphatic metastasis of CD133+ hepatocarcinoma ascites syngeneic cell lines mediated by JNK signaling pathway in vitro and in vivo. Biomed Pharmacother. 2013;67:337–45.CrossRefPubMedGoogle Scholar
  30. 30.
    Hashiguchi M, Ueno S, Sakoda M, Iino S, Hiwatashi K, Minami K, et al. Clinical implication of ZEB-1 and E-cadherin expression in hepatocellular carcinoma (HCC). BMC Cancer. 2013;13:572.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Natsuizaka M, Omura T, Akaike T, Kuwata Y, Yamazaki K, Sato T, et al. Clinical features of hepatocellular carcinoma with extrahepatic metastases. J Gastroenterol Hepatol. 2005;20:1781–7.CrossRefPubMedGoogle Scholar
  32. 32.
    Wang JL, Gray RM, Haudek KC, Patterson RJ. Nucleocytoplasmic lectins. Biochim et Biophys Acta (BBA)-Gen Sub. 2004;1673:75–93.CrossRefGoogle Scholar
  33. 33.
    Chiu J, Wong JW, Gerometta M, Hogg PJ. Mechanism of dimerization of a recombinant mature vascular endothelial growth factor C. Biochemistry. 2014;53:7–9.CrossRefPubMedGoogle Scholar
  34. 34.
    Pin AL, Houle F, Fournier P, Guillonneau M, Paquet ER, Simard MJ, et al. Annexin-1-mediated endothelial cell migration and angiogenesis are regulated by vascular endothelial growth factor (VEGF)-induced inhibition of miR-196a expression. J Biol Chem. 2012;287:30541–51.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Tsurusaki T, Kanda S, Sakai H, Kanetake H, Saito Y, Alitalo K, et al. Vascular endothelial growth factor-C expression in human prostatic carcinoma and its relationship to lymph node metastasis. Br J Cancer. 1999;80:309–13.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Skobe M, Hawighorst T, Jackson DG, Prevo R, Janes L, Velasco P, et al. Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis. Nat Med. 2001;7:192–8.CrossRefPubMedGoogle Scholar
  37. 37.
    Addya S, Shiroto K, Turoczi T, Zhan L, Kaga S, Fukuda S, et al. Ischemic preconditioning-mediated cardioprotection is disrupted in heterozygous Flt-1 (VEGFR-1) knockout mice. J Mol Cell Cardiol. 2005;38:345–51.CrossRefPubMedGoogle Scholar
  38. 38.
    Rozanov DV, Savinov AY, Williams R, Liu K, Golubkov VS, Krajewski S, et al. Molecular signature of MT1-MMP: transactivation of the downstream universal gene network in cancer. Cancer Res. 2008;68:4086–96.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Torosyan Y, Simakova O, Naga S, Mezhevaya K, Leighton X, Diaz J, et al. Annexin-A7 protects normal prostate cells and induces distinct patterns of RB-associated cytotoxicity in androgen-sensitive and -resistant prostate cancer cells. Int J Cancer. 2009;125:2528–39.CrossRefPubMedGoogle Scholar
  40. 40.
    Srivastava M, Torosyan Y, Raffeld M, Eidelman O, Pollard HB, Bubendorf L. ANXA7 expression represents hormone-relevant tumor suppression in different cancers. Int J Cancer. 2007;121:2628–36.CrossRefPubMedGoogle Scholar
  41. 41.
    Leighton X, Srikantan V, Pollard HB, Sukumar S, Srivastava M. Significant allelic loss of ANX7region (10q21) in hormone receptor negative breast carcinomas. Cancer Lett. 2004;210:239–44.CrossRefPubMedGoogle Scholar
  42. 42.
    Srivastava M, Montagna C, Leighton X, Glasman M, Naga S, Eidelman O, et al. Haploinsufficiency of Anx7 tumor suppressor gene and consequent genomic instability promotes tumorigenesis in the Anx7(+/-) mouse. Proc Natl Acad Sci U S A. 2003;100:14287–92.CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Srivastava M, Bubendorf L, Raffeld M, Bucher C, Torhorst J, Sauter G, et al. Prognostic impact of ANX7-GTPase in metastatic and HER2-negative breast cancer patients. Clin Cancer Res. 2004;10:2344–50.CrossRefPubMedGoogle Scholar
  44. 44.
    Wu B, Zhang F, Yu M, Zhao P, Ji W, Zhang H, et al. Up-regulation of Anxa2 gene promotes proliferation and invasion of breast cancer MCF-7 cells. Cell Prolif. 2012;45:189–98.CrossRefPubMedGoogle Scholar
  45. 45.
    Sun Y, Gao G, Cai J, Wang Y, Qu X, He L, et al. Annexin A2 is a discriminative serological candidate in early hepatocellular carcinoma. Carcinogenesis. 2013;34:595–604.CrossRefPubMedGoogle Scholar
  46. 46.
    Jeon YR, Kim SY, Lee EJ, Kim YN, Noh DY, Park SY, et al. Identification of annexin II as a novel secretory biomarker for breast cancer. Proteomics. 2013;13:3145–56.CrossRefPubMedGoogle Scholar
  47. 47.
    Li X, Chen L, Liang XJ, Gao YF, Wang XJ, Xu Q, et al. Annexin A5 protein expression is associated with the histological differentiation of uterine cervical squamous cell carcinoma in patients with an increased serum concentration. Mol Med Rep. 2012;6:1249–54.PubMedGoogle Scholar
  48. 48.
    Huang Y, Du Y, Zhang X, Bai L, Mibrahim M, Zhang J, et al. Down-regulated expression of Annexin A7 induces apoptosis in mouse hepatocarcinoma cell line by the intrinsic mitochondrial pathway. Biomedicine & Pharmacotherapy. 2015.Google Scholar
  49. 49.
    Smitherman AB, Mohler JL, Maygarden SJ, Ornstein DK. Expression of annexin I, II and VII proteins in androgen stimulated and recurrent prostate cancer. J Urol. 2004;171:916–20.CrossRefPubMedGoogle Scholar
  50. 50.
    Qazi AS, Sun M, Huang Y, Wei Y, Tang J. Subcellular proteomics: determination of specific location and expression levels of lymphatic metastasis associated proteins in hepatocellular carcinoma by subcellular fractionation. Biomed Pharmacother. 2011;65:407–16.CrossRefPubMedGoogle Scholar
  51. 51.
    Zhang Y, Meng X, Zeng H, Guan Y, Zhang Q, Guo S, et al. Serum vascular endothelial growth factor-C levels: A possible diagnostic marker for lymph node metastasis in patients with primary non-small cell lung cancer. Oncol Lett. 2013;6:545–9.PubMedPubMedCentralGoogle Scholar
  52. 52.
    Mukozu T, Nagai H, Matsui D, Kanekawa T, Sumino Y. Serum VEGF as a tumor marker in patients with HCV-related liver cirrhosis and hepatocellular carcinoma. Anticancer Res. 2013;33:1013–21.PubMedGoogle Scholar
  53. 53.
    Duan L, Ye L, Zhao G, Wu Z, Jin C, Cai X, et al. Serum spleen tyrosine kinase and vascular endothelial growth factor-C levels predict lymph node metastasis of oesophageal squamous cell carcinoma. Eur J Cardiothorac Surg. 2013;43:e58–63.CrossRefPubMedGoogle Scholar
  54. 54.
    Wang TB, Wang J, Wei XQ, Wei B, Dong WG. Serum vascular endothelial growth factor-C combined with multi-detector CT in the preoperative diagnosis of lymph node metastasis of gastric cancer. Asia Pac J Clin Oncol. 2012;8:180–6.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  • Xian-Yan Wang
    • 1
  • Feng Gao
    • 2
  • Yu-Rong Sun
    • 1
  • Lu-Lu Bai
    • 3
  • Mohammed Mohammed Ibrahim
    • 3
  • Bo Wang
    • 3
  • Jian-Wu Tang
    • 3
  1. 1.Department of PathologyQiqihar Medical UniversityQiqiharPeople’s Republic of China
  2. 2.Department of AnesthesiaThe Third Affiliated Hospital of Qiqihar Medical UniversityQiqiharPeople’s Republic of China
  3. 3.Key Laboratory of Tumor Metastasis of Liaoning Province, Department of PathologyDalian Medical UniversityDalianPeople’s Republic of China

Personalised recommendations