DICKKOPF-4 activates the noncanonical c-Jun-NH2 kinase signaling pathway while inhibiting the Wnt-canonical pathway in human renal cell carcinoma

Abstract

Background: To the authors' knowledge, the functional significance of the Wnt antagonist dickkopf homolog 4 (DKK4) has not been investigated previously in renal cancer.

Methods: The authors initially observed that the expression of DKK4 was significantly higher in renal cancer tissues compared with adjacent normal kidney tissues. To assess the function of DKK4, stable DKK4-transfected cells were established, and functional analyses were performed, including a T-cell factor/lymphoid enhancer factor (TCF/LEF) reporter assay and tests for cell viability, colony formation, apoptosis, cell cycle, invasive capability, wound-healing capability, and in vivo tumor growth.

Results: The relative TCF/LEF activity was significantly lower in DKK4-transfected cells compared with empty vector, and nuclear β-catenin expression was decreased in DKK4 transfectants. In addition, expression levels of the β-catenin downstream effector proteins cyclin D1 and c-Myc were decreased in DKK4 transfectants. However, greater invasiveness and migration were observed in stably transfected DKK4 cells. Increased growth of DKK4-transfected tumors also was observed in nude mice. Members of the Wnt noncanonical/c-Jun-NH2 kinase (JNK) signaling pathway also were effected, such as c-Jun, which had significantly increased expression and phosphorylation in DKK4-stable transfectants, and matrix metalloproteinase-2, which had significantly increased expression in DKK4-stable transfectants.

Conclusions: This is the first study to indicate that DKK4 expression is increased in renal cancer tissues and that DKK4 activates the noncanonical JNK signaling pathway while inhibiting the Wnt-canonical pathway.

Figure 1. DKK4 mRNA expression level in human renal cancer tissues and adjacent normal kidney tissues (n= 30)

Expression levels of DKK4 in renal cancer tissues and adjacent normal kidney tissues (30 clear cell carcinoma patients). When the relative DKK4 mRNA expression (DKK4/GAPDH) was higher in renal cancer tissues compared to matched normal renal tissues, the case was included in the category where “the DKK4 expression high in cancer tissues”.

Figure 2. TCF/LEF reporter assay, establishment of DKK4 transfectants (transient, stable), cell viability and colony formation assay in DKK4 transfectants

A. TCF/LEF reporter assay. (72 hours transient transfection) B. Expression of DKK4 in stable transfectant (A-498 cells) and transient transfectant (Caki-1 cells) by real-time RT PCR and Western blots. C. Cell viability assay in empty vector and DKK4 transfected cells (A-498-clone 1, clone 2; Caki-1) (MTS assay). D. colony formation assay in empty vector and stably DKK4 transfected cells (clone 1, clone 2).

Figure 3. Invasion, wound healing assay and in vivo study with empty vector and DKK4 transfected renal cancer cells

A. Invasion assay, B. Wound healing assay (A-498 cells-0/ 10 hours; Caki-1 cells-0/8 hours), C. Assessment of promotion of tumor growth in nude mice with empty vector and DKK4 clone 1 and clone 2 cells (C-1). Representative picture of nude mice with DKK4 transfected and empty vector cells are shown (C-2). DKK4 protein expression was confirmed by immunohistochemistry (C-2).

Figure 4. Apoptosis assay with stably (A-498) or transient (Caki-1) transfected empty and DKK4 renal cancer cells and protein expression of apoptosis markers

A. Flow cytometry analysis of apoptosis in empty vector and stable DKK4 clone 1 and clone 2 cells and transient DKK4 transfectants of Caki-1 cells. Annexin V-FITC and 7-AAD were measured by flow cytometry. Representative results are also shown (B). Data are the mean ± S.D. of four independent experiments. C. Protein expression of pro-caspase-3 and cleaved caspase3 in empty and DKK4 stable transfectants (A-498) and DKK4 transient transfectants (Caki-1).

Figure 5. Protein expression of signaling pathway genes in empty vector and stable DKK4 transfected A-498 Cells

A. Wnt-canonical pathway related genes expression (Western blot assay). B. Beta-catenin expression in cytoplasm and nuclei. C. Non-canonical pathway gene expression (Western blot assay)