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Oncol Rep. 29(4):1349-55. 2013. doi: 10.3892/or.2013.2251.  

Knockdown of Dkk-3 decreases cancer cell migration and invasion independently of the Wnt pathways in oral squamous cell carcinoma‑derived cells.

Katase NLefeuvre MTsujigiwa HFujii MIto STamamura RBuery RRGunduz MNagatsuka H.


Oral squamous cell carcinoma (OSCC) is thought to arise as the result of cumulative genetic or epigenetic alterations in cancer-associated genes. We focused on the Dickkopf-3 (Dkk-3) gene as a candidate tumor suppressor in OSCC. Dkk-3 is a potential tumor suppressor, and its downregulation has been reported in various types of malignancies. However, our previous data demonstrated that the Dkk-3 protein was dominantly expressed in OSCC tissue, and its expression was correlated with a high incidence of metastasis and with poor prognosis. In order to explain this paradox, we performed functional analyses of the Dkk-3 gene in cancer cell lines. RT-PCR revealed that Dkk-3 mRNA expression was observed in OSCC-derived cell lines but not in gastrointestinal or colorectal adenocarcinoma‑derived cell lines. The siRNA for Dkk-3 was transfected into Dkk-3-expressing cells, and the changes in cell proliferation, invasion and migration were assessed. The knockdown of Dkk-3 mRNA by siRNA transfection did not affect cell proliferation, but it significantly decreased cell migration and invasion. To further investigate the precise mechanism that contributes to the potential oncogenic function of Dkk-3, the Wnt canonical pathway and non-canonical pathways were assessed. Western blotting demonstrated that the effect of Dkk-3 knockdown on cell migration or invasion was not caused by activation of the Wnt pathways. These data demonstrated that Dkk-3 expression in OSCC was different than that in adenocarcinomas. Dkk-3 may possess an oncogenic function that is independent of Wnt signaling.


What to examine:What would be conduced when DKK3 gene was silenced by siRNA in HNSCC cells? 

Results:DKK3 silencing by siRNA resulted in significantly reduced cellular migration invasion by certain signaling pathway

              other than Wnt signaling pathway.

Previously, I examined DKK3 protein expression in HNSCC tissue samples and shown that unlike other cancer type, HNSCC cells express DKK3 protein, and that patients with DKK3  expression showed shorter disease free-survival and metastasis free survival.

​These results implies that DKK3 specifically exert oncogenic function in HNSCC. In the present study, "loss-of-function" experiment was performed in HNSCC cell lines (Ca9-22 and HSC-4)


(1)Transient knockdown of DKK3 by transfection of siRNA

  siRNAs targeting DKK3 were transfected into HNSCC-derived cells, Ca9-22 and HSC-4.

  In both cells, siRNA successfully reduced DKK3 mRNA expression.

  The effects of this DKK3 transient knockdown were investigated.

(2)DKK3 transient knockdown resulted in reduced cellular migration and invasion, but

        did not affect on cellular proliferation.

  In both of cell lines, DKK3 transient knockdown reduced cellular migration and invasion.






(3)Reduction of cellular migration and invasion were not driven by Wnt signaling pathway.

  Western blotting revealed that this reduced cellular migration and invasion was

  not caused by Wnt signaling.

  From all the data, I concluded that  "Knockdown of DKK3 in HNSCC cells

  reduced cellular migration and invasion independently from

  Wnt signaling pathway."

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