Oral Dis. 2022 [in press]

DKK3/CKAP4 axis is associated with advanced stage and poorer prognosis

in oral cancer

 

Katase N, Kudo K, Ogawa K, Sakamto Y, Nishimatsu S, Yamauchi A, 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.

<研究の概要>

検証項目:食道扁平上皮癌でDKK3の受容体として機能するCKAP4のHNSCCでの発現と機能は?DKK3/CKAP4は治療ターゲットなるか?

結果:HNSCCでもDKK3/CKAP4発現は高く、予後不良因子であった。さらにDKK3/CKAP4に対する抗体は腫瘍細胞の増殖・浸潤・遊走を全て有意に低下させた(= DKK3/CKAP4 axisはHNSCC治療の有望なターゲットである)。

 

前回の論文(Katase N et al. Int J Oncol. 2019)では

頭頸部扁平上皮癌(HNSCC)細胞ではDKK3がAktの活性化を介して

腫瘍細胞の悪性度を規定することを明らかにした。

その過程では、DKK3が何らかの受容体を介してAktを活性化する

ことが示唆されていたが、DKK3の受容体はこれまで報告がなかった。

 

2018年にKajiwaraらが同じくDKK3を高発現する食道扁平上皮癌に

おいてCKAP4がDKK3の受容体として機能することを報告したことを

受け、本研究ではHNSCCでのDKK3/CKAP4発現と、DKK3/CKAP4

axisの抗体による阻害が治療に有効かを検討した。

(1) HNSCCでのDKK3, CKAP4発現

 免疫染色でDKK3/CKAP4発現を検討し、臨床データとの相関を検討した。

 DKK3はoverall survivalに対する予後不良因子、CKAP4はdisease-free

    survivalに対する予後不良因子であることが示された。

 

 

(2) 抗体によるDKK3, CKAP4阻害の影響

 DKK3/CKAP4に対する抗体をHNSCC細胞に投与すると、Aktのリン酸化と

 ともに細胞の増殖、浸潤、遊走が全て有意に抑制された。

 

以上から、DKK3/CKAP4はHNSCC治療の有望なターゲットとなる可能性が示唆された

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