DKK3 knockdown confers negative effects on the malignant potency of head and neck squamous cell carcinoma cells via the PI3K/Akt and MAPK signaling pathways

Int J Oncol. 2019 Mar;54(3):1021-1032. doi: 10.3892/ijo.2018.4667. Epub 2018 Dec 14.

Abstract

Dickkopf‑related protein 3 (DKK3), which is a member of the Dickkopf WNT signaling pathway inhibitor family, is considered to be a tumor suppressor, due to its reduced expression in cancer cells and its ability to induce apoptosis when overexpressed by adenovirus. However, our previous study demonstrated alternative functions for DKK3 in head and neck squamous cell carcinoma (HNSCC). Our study reported that DKK3 expression was predominantly upregulated in HNSCC cell lines and tissue samples, and its expression was significantly correlated with poor prognosis. Furthermore, DKK3 overexpression in HNSCC cells significantly increased cancer cell proliferation, migration, invasion and in vivo tumor growth. These data have led to the hypothesis that DKK3 may exert oncogenic functions and may increase the malignant properties of HNSCC. The present study established a stable DKK3 knockdown cell line (HSC‑3 shDKK3) using lentivirus‑mediated short hairpin RNA, and assessed its effects on cancer cell behavior using MTT, migration and invasion assays. In addition, its effects on in vivo tumor growth were assessed using a xenograft model. Furthermore, the molecular mechanisms underlying the effects of DKK3 knockdown were investigated by microarray analysis, pathway analysis and western blotting. Compared with control cells, HSC‑3 shDKK3 cells exhibited significantly reduced proliferation, migration and invasion, and formed significantly smaller tumor masses when subcutaneously transplanted into nude mice. In addition, in HSC‑3 shDKK3 cells, the expression levels of phosphorylated (p)‑protein kinase B (Akt) (Ser473), p‑phosphoinositide 3‑kinase (PI3K) p85 (Tyr467), p‑PI3K p55 (Try199), p‑3‑phosphoinositide‑dependent protein kinase‑1 (PDK1) (Ser241) and total p38 mitogen‑activated protein kinase (MAPK) were reduced. Furthermore, phosphorylation of mechanistic target of rapamycin (mTOR) (Ser2448) was slightly decreased in HSC‑3 shDKK3 cells, which may be due to the increased expression of DEP domain‑containing mTOR‑interacting protein. Conversely, DKK3 overexpression in HSC‑3 shDKK3 cells rescued cellular proliferation, migration and invasion. With regards to expression levels, p‑PI3K and p‑PDK1 expression was not altered, whereas mTOR and p‑p38 MAPK expression was elevated. These data supported the hypothesis and indicated that DKK3 may contribute to the malignant phenotype of HNSCC cells via the PI3K/Akt/mTOR and MAPK signaling pathways.

Keywords: head and neck squamous cell carcinoma; Dickkopfrelated protein 3; phosphoinositide 3-kinase; protein kinase B; mitogen-activated protein kinase.

Publication types

  • Comparative Study

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Animals
  • Cell Line, Tumor
  • Cell Movement
  • Cell Proliferation
  • Chemokines
  • Computational Biology
  • Gene Knockdown Techniques
  • Humans
  • Intercellular Signaling Peptides and Proteins / deficiency
  • Intercellular Signaling Peptides and Proteins / genetics*
  • Intercellular Signaling Peptides and Proteins / metabolism*
  • MAP Kinase Signaling System* / genetics
  • Mice, Nude
  • Microarray Analysis
  • Neoplasm Invasiveness
  • Phosphatidylinositol 3-Kinases / genetics
  • Phosphatidylinositol 3-Kinases / metabolism*
  • Prognosis
  • Proto-Oncogene Proteins c-akt / genetics
  • Proto-Oncogene Proteins c-akt / metabolism*
  • Signal Transduction / genetics
  • Squamous Cell Carcinoma of Head and Neck / genetics
  • Squamous Cell Carcinoma of Head and Neck / metabolism
  • Squamous Cell Carcinoma of Head and Neck / physiopathology*
  • TOR Serine-Threonine Kinases / metabolism

Substances

  • Adaptor Proteins, Signal Transducing
  • Chemokines
  • DKK3 protein, human
  • Intercellular Signaling Peptides and Proteins
  • MTOR protein, human
  • Proto-Oncogene Proteins c-akt
  • TOR Serine-Threonine Kinases