Tumor Biology

, Volume 37, Issue 8, pp 10827–10838 | Cite as

Transcriptomic changes associated with DKK4 overexpression in pancreatic cancer cells detected by RNA-Seq

Original Article


The promotion of tumor development by Dickkopf 4 (DKK4) is receiving increased attention. However, the association between DKK4 and pancreatic cancer remains unclear. DKK4 expression was measured in pancreatic ductal adenocarcinoma tissues using qRT-PCR and immunohistochemistry. A DKK4-overexpressing pancreatic cancer cell line was established, and the differentially expressed genes (DEGs) that were induced by DKK4 were identified using transcriptome sequencing. The association between the identified DEGs and pancreatic cancer was assessed using gene ontology (GO), pathway analysis, pathway interaction networks, differentially expressed gene interaction network analysis, and co-expression gene networks. Finally, the accuracy of the analyses was validated using serial paraffin and frozen sections of clinical samples. DKK4 is highly expressed in pancreatic cancer tissues. DEGs of overexpression DKK4 of PANC-1 are mostly upregulated. GO and pathway analysis showed that DKK4 are associated with tumor and organ development and immune inflammation. The mitogen-activated protein kinase (MAPK) signaling pathway was the main signal transduction pathway that showed significant enrichment in overexpression DKK4 of PANC-1. The results of GO, pathway analyses, and differentially expressed gene interaction network identified genes that are closely associated with tumor development, including MAPK3, PIK3R3, VAV3, JAG1, and Notch3. The immunohistochemistry and immunofluorescence results suggested that DKK4 is co-expressed with MAPK3 and VAV3 in pancreatic cancer tissues. The results presented here show for the first time that DKK4 is highly expressed in pancreatic cancer tissues. Bioinformatics analysis of a DKK4-overexpressing of PANC-1 identified several oncogenes that are closely associated with tumors, and the MAPK signaling pathway is the core signal transduction pathway. DKK4 can be co-expressed with MAPK3 and VAV3 in pancreatic ductal adenocarcinoma tissues. Thus, DKK4 may have function on the development and progression of pancreatic cancer.


DKK4 Pancreatic cancer RNA sequencing Transcriptome Oncogenes 


Compliance with ethical standards

Conflict of interest


Financial support

This paper is supported by the following grants: National Natural Science Foundation of China (No. 81072439, the National High Technology Research and Development Program of China (863 Program) (No. 2012AA021105), and the Research Special Fund for Public Welfare Industry of Health (No. 201202007). There has been no industrial or pharmaceutical support.

Supplementary material

13277_2015_4379_Fig7_ESM.gif (15 kb)

(GIF 15 kb)

13277_2015_4379_MOESM1_ESM.tif (20 kb)
High Resolution (TIF 20 kb)
13277_2015_4379_Fig8_ESM.gif (18 kb)

(GIF 18 kb)

13277_2015_4379_MOESM2_ESM.tif (22 kb)
High Resolution (TIF 21 kb)
13277_2015_4379_Fig9_ESM.gif (48 kb)

(GIF 47 kb)

13277_2015_4379_MOESM3_ESM.tif (22.4 mb)
High Resolution (TIF 22,919 kb)
13277_2015_4379_Fig10_ESM.gif (102 kb)

(GIF 102 kb)

13277_2015_4379_MOESM4_ESM.tif (26.3 mb)
High Resolution (TIF 26,923 kb)
13277_2015_4379_MOESM5_ESM.docx (16 kb)
Supplementary Table 1 (DOCX 15 kb)
13277_2015_4379_MOESM6_ESM.xls (1.9 mb)
Supplementary Table 2 (XLS 1995 kb)
13277_2015_4379_MOESM7_ESM.xls (1.5 mb)
Supplementary Table 3 (XLS 1495 kb)


  1. 1.
    Ben Q, Wang K, Yuan Y, Li Z. Pancreatic cancer incidence and outcome in relation to ABO blood groups among Han Chinese patients: a case-control study. Int J Cancer. 2011;128:1179–86.CrossRefPubMedGoogle Scholar
  2. 2.
    Polakis P. The many ways of Wnt in cancer. Curr Opin Genet Dev. 2007;17:45–51.CrossRefPubMedGoogle Scholar
  3. 3.
    Grotewold L, Ruther U. The Wnt antagonist Dickkopf-1 is regulated by Bmp signaling and c-Jun and modulates programmed cell death. EMBO J. 2002;21:966–75.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Guder C, Pinho S, Nacak TG, Schmidt HA, Hobmayer B, Niehrs C, et al. An ancient Wnt-Dickkopf antagonism in Hydra. Development. 2006;133:901–11.CrossRefPubMedGoogle Scholar
  5. 5.
    Zhai W, Hu GH, Zheng JH, Peng B, Liu M, Huang JH, et al. High expression of the secreted protein dickkopf homolog 4: roles in invasion and metastasis of renal cell carcinoma and its association with Von Hippel-Lindau gene. Int J Mol Med. 2014;33:1319–26.PubMedGoogle Scholar
  6. 6.
    Matsui A, Yamaguchi T, Maekawa S, Miyazaki C, Takano S, Uetake T, et al. DICKKOPF-4 and -2 genes are upregulated in human colorectal cancer. Cancer Sci. 2009;100:1923–30.CrossRefPubMedGoogle Scholar
  7. 7.
    Pendas-Franco N et al. DICKKOPF-4 is induced by TCF/beta-catenin and upregulated in human colon cancer, promotes tumour cell invasion and angiogenesis and is repressed by 1 alpha, 25-dihydroxyvitamin D-3. Oncogene. 2008;27(32):4467–77.CrossRefPubMedGoogle Scholar
  8. 8.
    Giovannetti E, Codacci-Pisanelli G, Peters GJ. TFAP2E-DKK4 and chemoresistance in colorectal cancer. N Engl J Med. 2012;366:966.CrossRefPubMedGoogle Scholar
  9. 9.
    Fatima S, Lee NP, Luk JM. Dickkopfs and Wnt/beta-catenin signalling in liver cancer. World J Clin Oncol. 2011;2:311–25.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Niehrs C. Function and biological roles of the Dickkopf family of Wnt modulators. Oncogene. 2006;25:7469–81.CrossRefPubMedGoogle Scholar
  11. 11.
    Yu B, Yang X, Xu Y, Yao G, Shu H, Lin B, et al. Elevated expression of DKK1 is associated with cytoplasmic/nuclear beta-catenin accumulation and poor prognosis in hepatocellular carcinomas. J Hepatol. 2009;50:948–57.CrossRefPubMedGoogle Scholar
  12. 12.
    Marioni JC, Mason CE, Mane SM, Stephens M, Gilad Y. RNA-seq: an assessment of technical reproducibility and comparison with gene expression arrays. Genome Res. 2008;18:1509–17.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Mardis ER. The impact of next-generation sequencing technology on genetics. Trends Genet. 2008;24:133–41.CrossRefPubMedGoogle Scholar
  14. 14.
    Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet. 2009;10:57–63.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Dupuy D, Bertin N, Hidalgo CA, Venkatesan K, Tu D, Lee D, et al. Genome-scale analysis of in vivo spatiotemporal promoter activity in Caenorhabditis elegans. Nat Biotechnol. 2007;25:663–8.CrossRefPubMedGoogle Scholar
  16. 16.
    Kanehisa M, Goto S, Kawashima S, Okuno Y, Hattori M. The KEGG resource for deciphering the genome. Nucleic Acids Res. 2004;32:D277–80.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Yi M, Horton JD, Cohen JC, Hobbs HH, Stephens RM. WholePathwayScope: a comprehensive pathway-based analysis tool for high-throughput data. BMC Bioinforma. 2006;7:30.CrossRefGoogle Scholar
  18. 18.
    Draghici S, Khatri P, Tarca AL, Amin K, Done A, Voichita C, et al. A systems biology approach for pathway level analysis. Genome Res. 2007;17:1537–45.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000;28:27–30.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003;1:2498–504.CrossRefGoogle Scholar
  21. 21.
    Pujana MA, Han JD, Starita LM, Stevens KN, Tewari M, Ahn JS, et al. Network modeling links breast cancer susceptibility and centrosome dysfunction. Nat Genet. 2007;39:1338–49.CrossRefPubMedGoogle Scholar
  22. 22.
    Prieto C, Risueño A, Fontanillo C, De las Rivas J. Human gene coexpression landscape: confident network derived from tissue transcriptomic profiles. Plos One. 2008;3:e3911.Google Scholar
  23. 23.
    Barabasi AL, Oltvai ZN. Network biology: understanding the cell’s functional organization. Nat Rev Genet. 2004;5:101–U15.CrossRefPubMedGoogle Scholar
  24. 24.
    Ravasz E, Somera AL, Mongru DA, Oltvai ZN, Barabási AL. Hierarchical organization of modularity in metabolic networks. Science. 2002;297:1551–5.CrossRefPubMedGoogle Scholar
  25. 25.
    Carlson MR, Zhang B, Fang Z, Mischel PS, Horvath S, Nelson SF. Gene connectivity, function, and sequence conservation: predictions from modular yeast co-expression networks. BMC Genomics. 2006;7:40.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Hirata H, Hinoda Y, Majid S, Chen Y, Zaman MS, Ueno K, et al. DICKKOPF-4 activates the noncanonical c-Jun-NH2 kinase signaling pathway while inhibiting the Wnt-canonical pathway in human renal cell carcinoma. Cancer. 2011;117:1649–60.CrossRefPubMedGoogle Scholar
  27. 27.
    Takahashi N, Fukushima T, Yorita K, Tanaka H, Chijiiwa K, Kataoka H. Dickkopf-1 is overexpressed in human pancreatic ductal adenocarcinoma cells and is involved in invasive growth. Int J Cancer. 2010;126:1611–20.PubMedGoogle Scholar
  28. 28.
    Yamamoto S, Tomita Y, Hoshida Y, Takiguchi S, Fujiwara Y, Yasuda T, et al. Expression of hepatoma-derived growth factor is correlated watch lymph node metastasis and prognosis of gastric carcinoma. Clin Cancer Res. 2006;12:117–22.CrossRefPubMedGoogle Scholar
  29. 29.
    Hu TH, Huang CC, Liu LF, Lin PR, Liu SY, Chang HW, et al. Expression of hepatoma-derived growth factor in hepatocellular carcinoma—a novel prognostic factor. Cancer. 2003;98:1444–56.CrossRefPubMedGoogle Scholar
  30. 30.
    Matsuyama A, Inoue H, Shibuta K, Tanaka Y, Barnard GF, Sugimachi K, et al. Hepatoma-derived growth factor is associated with reduced sensitivity to irradiation in esophageal cancer. Cancer Res. 2001;61:5714–7.PubMedGoogle Scholar
  31. 31.
    Mao J, Xu Z, Fang Y, Wang H, Xu J, Ye J, et al. Hepatoma-derived growth factor involved in the carcinogenesis of gastric epithelial cells through promotion of cell proliferation by Erk1/2 activation. Cancer Sci. 2008;99:2120–7.CrossRefPubMedGoogle Scholar
  32. 32.
    Tang Y, Liu F, Zheng C, Sun S, Jiang Y. Knockdown of clusterin sensitizes pancreatic cancer cells to gemcitabine chemotherapy by ERK1/2 inactivation. J Exp Clin Cancer Res. 2012;31:73.CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Deng Y, Wang J, Wang G, Jin Y, Luo X, Xia X, et al. p55PIK transcriptionally activated by MZF1 promotes colorectal cancer cell proliferation. Biomed Res Int. 2013;2013:868131.PubMedGoogle Scholar
  34. 34.
    Soroceanu L, Kharbanda S, Chen R, Soriano RH, Aldape K, Misra A, et al. Identification of IGF2 signaling through phosphoinositide-3-kinase regulatory subunit 3 as a growth-promoting axis in glioblastoma. Proc Natl Acad Sci U S A. 2007;104:3466–71.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Pande M, Thompson PA, Do KA, Sahin AA, Amos CI, Frazier ML, et al. Genetic variants in the vitamin D pathway and breast cancer disease-free survival. Carcinogenesis. 2013;34:587–94.CrossRefPubMedGoogle Scholar
  36. 36.
    Ezzeldin M, Borrego-Diaz E, Taha M, Esfandyari T, Wise AL, Peng W, et al. RalA signaling pathway as a therapeutic target in hepatocellular carcinoma (HCC). Mol Oncol. 2014;8:1043–53.CrossRefPubMedGoogle Scholar
  37. 37.
    Tao T, Cheng C, Ji Y, Xu G, Zhang J, Zhang L, et al. Numbl inhibits glioma cell migration and invasion by suppressing TRAF5-mediated NF-kappaB activation. Mol Biol Cell. 2012;23:2635–44.CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Wang J, Yang H, Li W, Xu H, Yang X, Gan L. Thioredoxin 1 upregulates FOXO1 transcriptional activity in drug resistance in ovarian cancer cells. Biochim Biophys Acta. 1852;2015:395–405.Google Scholar
  39. 39.
    Javidi-Sharifi N, Traer E, Martinez J, Gupta A, Taguchi T, Dunlap J, et al. Crosstalk between KIT and FGFR3 promotes gastrointestinal stromal tumor cell growth and drug resistance. Cancer Res. 2015;75:880–91.CrossRefPubMedGoogle Scholar
  40. 40.
    Li ZP, Li X, Yu C, Wang M, Peng F, Xiao J, et al. MicroRNA-100 regulates pancreatic cancer cells growth and sensitivity to chemotherapy through targeting FGFR3. Tumour Biol. 2014;35:11751–9.CrossRefPubMedGoogle Scholar
  41. 41.
    Bertagnolo V, Benedusi M, Brugnoli F, Lanuti P, Marchisio M, Querzoli P, et al. Phospholipase C-beta 2 promotes mitosis and migration of human breast cancer-derived cells. Carcinogenesis. 2007;28:1638–45.CrossRefPubMedGoogle Scholar
  42. 42.
    Zhang B, Horvath S. A general framework for weighted gene co-expression network analysis. Stat Appl Genet Mol Biol. 2005;4:Article 17.Google Scholar
  43. 43.
    Horvath S, Zhang B, Carlson M, Lu KV, Zhu S, Felciano RM, et al. Analysis of oncogenic signaling networks in glioblastoma identifies ASPM as a molecular target. Proc Natl Acad Sci U S A. 2006;103:17402–7.CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    He DN, Liu ZP, Honda M, Kaneko S, Chen L. Coexpression network analysis in chronic hepatitis B and C hepatic lesions reveals distinct patterns of disease progression to hepatocellular carcinoma. J Mol Cell Biol. 2012;4:140–52.CrossRefPubMedGoogle Scholar
  45. 45.
    Murat A, Migliavacca E, Gorlia T, Lambiv WL, Shay T, Hamou MF, et al. Stem cell-related “self-renewal” signature and high epidermal growth factor receptor expression associated with resistance to concomitant chemoradiotherapy in glioblastoma. J Clin Oncol. 2008;26:3015–24.CrossRefPubMedGoogle Scholar
  46. 46.
    Tan B, Li Y, Zhao Q, Fan L, Liu Y, Wang D, et al. Inhibition of Vav3 could reverse the drug resistance of gastric cancer cells by downregulating JNK signaling pathway. Cancer Gene Ther. 2014;21:526–31.CrossRefPubMedGoogle Scholar
  47. 47.
    Tan BB, Li Y, Zhao Q, Fan L, Wang D, Liu Y. Inhibition of gastric cancer cell growth and invasion through siRNA-mediated knockdown of guanine nucleotide exchange factor Vav3. Tumour Biol. 2014;35:1481–8.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2016

Authors and Affiliations

  1. 1.Institute of Hepatopancreatobiliary Surgery, Southwest HospitalThird Military Medical UniversityChongqingChina
  2. 2.Organ transplantation centreFirst Affiliated Hospital Sun Yat-sen UniversityGuangzhouChina
  3. 3.Translational Research Laboratory, Department of PathologyStony Brook UniversityStony BrookUSA

Personalised recommendations