Oncol Res. 26(1):45-58. 2018. doi: 10.3727/096504017X14926874596386.
DKK3 Overexpression Increases the Malignant Properties of Head and Neck Squamous Cell Carcinoma Cells.
Katase N, Nishimatsu SI, Yamauchi A, Yamamura M, Terada K, Itadani M, Okada N, Hassan NMM, Nagatsuka H,
Abstract
DKK3, a member of the dickkopf Wnt signaling pathway inhibitor family, is believed to be a tumor suppressor because of its reduced expression in cancer cells. However, our previous studies have revealed that DKK3 expression is predominantly observed in head and neck/oral squamous cell carcinoma (HNSCC/OSCC). Interestingly, HNSCC/OSCC patients with DKK3 expression showed a high rate of metastasis and poorer survival, and siRNA-mediated knockdown of DKK3 in HNSCC-derived cancer cell lines resulted in reduced cellular migration and invasion. From these data, it was hypothesized that DKK3 might exert an oncogenic function specific to HNSCC. In the present research, the DKK3 overexpression model was established, and its influences were investigated, together with molecular mechanism studies. The DKK3 expression profile in cancer cell lines was investigated, including HNSCC/OSCC, esophageal, gastric, colorectal, pancreatic, prostatic, and lung cancers. DKK3 overexpression was performed in HNSCC-derived cells by transfection of expression plasmid. The effects of DKK3 overexpression were assessed on cellular proliferation, migration, invasion, and in vivo tumor growth. The molecular mechanism of DKK3 overexpression was investigated by Western blotting and microarray analysis. DKK3 overexpression significantly elevated cellular proliferation, migration, and invasion, as well as increased mRNA expression of cyclin D1 and c-myc. However, reporter assays did not show TCF/LEF activation, suggesting that the increased malignant property of cancer cells was not driven by the Wnt/β-catenin pathway. For the investigation of the pathways/molecules in DKK3-mediated signals, the Western blot analyses revealed that phosphorylation of Akt (S473) and c-Jun (Ser63) was elevated. The application of a PI3K kinase inhibitor, LY294002, on HSC-3 DKK3 cells significantly decreased tumor cell proliferation, migration, and invasion. From these results, we demonstrated that DKK3 might contribute to cellular proliferation, invasion, migration, and tumor cell survival in HNSCC cells through a mechanism other than the canonical Wnt signaling pathway, which might be attributed to PI3K-Akt signaling.
＜Summary＞
What to examine：What would be conduced when DKK3 gene was overexpressed in HNSCC cells?
Results：DKK3 overexpression resulted in significantly elevated cellular proliferation, migration invasion and in vivo tumor growth (=malignant property of cancer cells) via phosphorylation of Akt(set473) and c-Jun.
In the previous article (Katase N et al. Oncol Rep. 2013), we have shown that knockdown of DKK3 by siRNA in HNSCC cell lines resulted in significantly reduced cellular migration and invasion by Wnt signal independent mechanism. In the present study, I performed "gain-of-function" experiment, aiming to investigate the effects of DKK3 overexpression in HNSCC cells.
(1) Overexpression of DKK3 in HNSCC-derived HSC-3 cell
Full-length DKK3 expressing plasmid was transfected into HSC-3,
a human tongue cancer-derived cells which express DKK3.
Significant increase in DKK3 protein/mRNA expression and secretion was confirmed.
(2) Effects of DKK3 overexpression in vitro/ in vivo
DKK3 overexpression resulted in significantly elevated cellular proliferation, migration,
and invasion. Moreover, DKK3 over expressed cells formed significantly larger tumor mass
with high Ki-67 index
(3) Why DKK3 increases malignant property of HNSCC cells?
DKK3 overexpression also elevated mRNA expression of
cyclin D1 and c-myc, both of which were target gene of Wnt
signaling. That disposed us as if "DKK3 might activate
Wnt signaling". However, reporter assay demonstrated that
there was no Wnt signal activation.
Series of Western blotting analyses revealed that
DKK3 overexpression induced phosphorylation of Akt(Ser473)
and c-Jun(Ser63).Application of Akt inhibitor, LY294002,
negated the effects of DKK3 overexpression.
From all the results obtained in this research, I concluded
that "DKK3 elevates malignant property of HNSCC
cells via Akt signaling".
