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 NNishimatsu SIYamauchi AYamamura MFujita 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.

<研究の概要>

検証項目:頭頸部扁平上皮癌細胞でDKK3遺伝子発現を安定的にノックダウンさせるとどうなるか?

結果:DKK3のstable knockdownでは、Akt, PDK1のリン酸化低下、p38MAPK発言低下を介して腫瘍細胞の増殖・浸潤・遊走・

   ヌードマウス皮下での腫瘍増殖が有意に低下した(=腫瘍細胞の悪性度が低下した)。

 

前回の論文(Katase N et al. Oncol Res. 2018)では、頭頸部扁平上皮癌(HNSCC)細胞にDKK3発現プラスミドをとランスフェクションすると

Akt, c-junのリン酸化上昇を介して腫瘍細胞の増殖・浸潤・遊走・ヌードマウス皮下での主要増殖が有意に増加することを示し、DKK3がHNSCC

細胞の悪性度を規定する可能性を報告した。今回は再度DKK3のLoss-of-function実験を計画し、HNSCC細胞にDKK3遺伝子に対するshRNAを

導入、DKK3 stable knock downモデルを構築してその影響を検討した。前回のGain-of-function実験と対になる実験である。

 

(1) HNSCC由来、HSC-3細胞でのDKK3 stable knockdown

DKK3遺伝子を発現しているHSC-3細胞に、lentivirus vector を用いてDKK3に

対するshRNAを導入、安定的にDKK3発現をノックダウンした細胞(HSC-3 shDKK3)を

作製した。コントロールとしてScramble配列を導入した細胞(HSC-3 shScr)を作製した。

DKK3発現が低下していることを確認し、MTT assay、migration assay、

invasion assayを行なった。

 

(2) in vitro/ in vivoでのDKK3 stable knockdownの影響

DKK3 stable knock downでは細胞増殖、遊走、浸潤の全てが有意に低下した。

ヌードマウス皮下への移植でも、DKK3 knock down細胞の腫瘍径は縮小し、

Ki-67 indexも有意に低かった。

 

 

 

 

 

(3) DKK3 knockdownで細胞の悪性度が低下するメカニズム

HSC-3、HSC-3 shScr、DKK3 shDKK3細胞RNAを抽出してmicroarray解析とpathway解析を行ったところ、PI3K/Akt/mTOR pathwayの

関与が示唆された。Western blottingでも、DKK3 knockdownによるPI3K, PDK1のリン酸化低下、p38MAPKの発現低下が認められた。

DKK3 knockdown細胞に再度DKK3発現プラスミドを導入すると細胞増殖・遊走・浸潤は全て回復し、Aktのリン酸化も増加した。

 

 

以上から、DKK3はHNSCCではAktを介して腫瘍の悪性度を規定する可能性がより強く示唆された

Oral pathology,  DKK3 functional analysis in

Head and neck squamous cell carcinoma

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