Department of Oral Pathology and Bone Metabolism, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.
Department of Molecular and Developmental Biology, Kawasaki Medical School, Kurashiki, Okayama, Japan.
Oncol Res. 2018 Jan 19;26(1):45-58. doi: 10.3727/096504017X14926874596386. Epub 2017 May 4.
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.
DKK3 是 Dickkopf Wnt 信号通路抑制剂家族的成员，由于其在癌细胞中的表达减少，被认为是一种肿瘤抑制因子。然而，我们之前的研究表明，DKK3 的表达主要在头颈部/口腔鳞状细胞癌 (HNSCC/OSCC) 中观察到。有趣的是，表达 DKK3 的 HNSCC/OSCC 患者转移率高，生存率差，而 HNSCC 衍生的癌细胞系中 DKK3 的 siRNA 介导敲低导致细胞迁移和侵袭减少。从这些数据中可以假设，DKK3 可能对头颈部特异性的肿瘤发挥致癌作用。在本研究中，建立了 DKK3 过表达模型，并进行了相关影响及其分子机制的研究。我们调查了包括 HNSCC/OSCC、食管癌、胃癌、结直肠癌、胰腺癌、前列腺癌和肺癌在内的多种癌细胞系中的 DKK3 表达谱。通过转染表达质粒，在 HNSCC 衍生细胞中进行 DKK3 过表达。评估 DKK3 过表达对细胞增殖、迁移、侵袭和体内肿瘤生长的影响。通过 Western blot 和微阵列分析研究 DKK3 过表达的分子机制。DKK3 过表达显著增加了细胞增殖、迁移和侵袭，并增加了 cyclin D1 和 c-myc 的 mRNA 表达。然而，报告基因检测未显示 TCF/LEF 激活，这表明癌细胞恶性程度的增加不是由 Wnt/β-catenin 通路驱动的。为了研究 DKK3 介导的信号通路/分子，Western blot 分析显示 Akt (S473) 和 c-Jun (Ser63) 的磷酸化水平升高。应用 PI3K 激酶抑制剂 LY294002 处理 HSC-3 DKK3 细胞后，肿瘤细胞的增殖、迁移和侵袭明显减少。由此，我们证明 DKK3 通过非经典 Wnt 信号通路以外的机制，可能通过 PI3K-Akt 信号通路，促进 HNSCC 细胞的细胞增殖、侵袭、迁移和肿瘤细胞存活。
