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.

<Summary>

What to examine:How is the expression and function of DKK3/CKAP4 in HNSCC?

                             Is application of antibodies against DKK3/CKAP4 effective for cancer suppression?

Results:DKK3/CKAP4 expression was high in HNSCC, and they are independent prognostic factor.

               Application of antibodies against DKK3/CKAP4 significantly suppressed cancer cell proliferation, migration and invasion.

 

 

We previously have shown that DKK3 exert oncogenic function via

activation of Akt signaling. (Katase N et al. Int J Oncol. 2019)

It is estimated that DKk3-medated Akt activation would be driven by

ligand-receptor interaction, but specific receptor for DKK3 has not been

identified for a long time,

In 2018, Kajiwara et al reported that CKAP4 functions as receptor for

DKK family proteins in esophageal squamous cell carcinoma.

Then, we investigated the expression of DKK3/CKAP4 in HNSCC, and

assessed whether antibodies against DKK3/CKAP4 could suppress

the cancer cells or not.

 

(1) DKK3/ CKAP4 expression in HNSCC

 Immunohistochemistry for DKK3/CKAP4 in HNSCC tissue samples

   revealed that the expression of these proteins are high, and associated

   with poorer prognosis.

 

 

(2) The effects of antibodies against DKK3/CKAP4

 Antibodies against DKK3/CKAP4 could significantly suppressed

   Akt phosphorylation, cellular proliferation, migration and invasion.

 

 

DKK3/CKAP4 axis would be the promising therapeutic target for HNSCC.

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