Int J Oncol. 2019 Mar;54(3):1021-1032. doi: 10.3892/ijo.2018.4667.
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.
Katase N, Nishimatsu SI, Yamauchi A, Yamamura M, Fujita S.
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.
＜Summary＞
What to examine：What would be conduced when DKK3 gene was stably knocked down in HNSCC cells?
Results：DKK3 stable knockdown resulted in significantly decreased cellular proliferation, migration invasion and in vivo tumor growth (=malignant property of cancer cells) via phosphorylation of PI3K/Akt/mTOR pathway.
In the previous article (Katase N et al. Oncol Res. 2018), we have shown that DKK3 overexpression in HNSCC cell lines resulted in significantly increased cellular proliferation, migration, invasion and in vivo tumor growth. It was hypothesized that DKK3 might modify the malignant property of the cancer cells. I the present study, we performed "loss-of-function" experiment.
(1) DKK3 stable knockdown
We established DKK3 stable knockdown cell from HNSCC-derived HSC-3 cells
(HSC-3 shDKK3), using lentivirus-mediated shRNA targeting for DKK3.
The effects of DKK3 stable knockdown was confirmed by MTT assay,
migration assay and invasion assay.
(2) The effects of DKK3 stable knockdown
DKK3 stable knock down resulted in significantly reduced celllar proliferation,
migration, invasion and in vivo tumor growth.
(3) The mechanism how DKK3 knockdown decreases malignant property of cancer cells
Microarray analysis with pathway analysis suggested involvement of PI3K/Akt/mTOR pathway in DKK3 knockdown cells.
Western blotting revealed that phosphorylation fof PI3K, PDK1 and expression of p38MAPK were decreased in HSC-3shDKK3.
Transfection of DKK3 expression plasmids into HSC-3 shDKK3 rescued cellular proliferation, migration and invasion.
From all the results obtained in this research, we concluded that "DKK3 regulates malignant property of HNSCC
cells via PI3K/Akt/mTOR signaling".
