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 NNishimatsu SIYamauchi AYamamura MTerada KItadani MOkada NHassan NMMNagatsuka H

Ikeda TNohno TFujita S.

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.

<研究の概要>

検証項目:頭頸部扁平上皮癌細胞でDKK3遺伝子発現を増加させるとどうなるか?

結果:DKK3過剰発現でAkt、c-Junのリン酸化が増加し腫瘍細胞の増殖・浸潤・遊走・ヌードマウス皮下での腫瘍増殖が有意に増大した

  (=腫瘍細胞の悪性度が増強された)

 

前回の論文(Katase N et al. Oncol Rep. 2013)では、頭頸部扁平上皮癌(HNSCC)細胞でDKK3遺伝子をsiRNAでノックダウンすると、Wnt signalとは無関係の機序で腫瘍の浸潤と遊走が有意に減少することを示した。今回はDKK3遺伝子機能解析のために、Gain-of-function実験としてHNSCC細胞にDKK3遺伝子を過剰発現させた場合の影響を検討した。前回のLoss-of-function実験と対になる実験である。

 

(1) HNSCC由来、HSC-3細胞への遺伝子過剰発現

すでにDKK3遺伝子を発現しているHSC-3細胞に、DKK3発現ベクターを

トランスフェクションして発現量を増大させた。DKK3発現が増大したことを確認し、

MTT assay、migration assay、invasion assayを行なった。

 

(2) in vitro/ in vivoでのDKK3過剰発現の影響

DKK3過剰発現では細胞増殖、遊走、浸潤の全てが有意に増大した。

ヌードマウス皮下への移植でも、DKK3過剰発現細胞は有意に大きい腫瘤を形成し、

Ki-67 indexも高かった。

 

 

 

 

 

 

 

 

 

 

 

 

(3) なぜDKK3過剰発現で細胞の悪性度が増加するのか

DKK3過剰発現細胞ではcyclin D1とc-myc遺伝子発現が上昇していた。

Cyclin D1、c-mycはともにWnt signalのターゲットであるので、

「DKK3がWnt signalを活性化しているのでは?」と考えた。

しかしreporter assayでWnt signalの活性化は否定された。

細胞増殖に関わる別のシグナルについてwestern blottingで

検討すると、Akt, c-junのリン酸化増加を見出した。

Aktの阻害剤であるLY294002をDKK3過剰発現細胞に加えると、

DKK3による細胞の悪性度増加は全て打ち消された。

 

 

 

 

以上から、「DKK3はHNSCCではAktを介して腫瘍の悪性度を高めている」と考えられた。

Oral pathology,  DKK3 functional analysis in

Head and neck squamous cell carcinoma

© 2018 by Naoki Katase. Proudly created with Wix.com

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