Expression of the Wnt antagonist Dickkopf-3 is associated with prognostic clinicopathologic characteristics and impairs proliferation and invasion in endometrial cancer

Abstract

Objective: Emerging evidence implicates the Wnt antagonist Dickkopf-3 (Dkk3) as a tumor suppressor and potential biomarker in solid tumors. We investigated whether Dkk3 plays an important role in the carcinogenesis of endometrial cancer (EC).

Methods: We analyzed Dkk3 mRNA expression via real-time RT-PCR in twenty-seven human primary EC tissues, and six matched normal endometrial controls. Dkk3 levels were correlated with various clinicopathologic characteristics. Additionally, enforced Dkk3 expression was examined in proliferation and tumorigenesis in vitro and in vivo, using MTT, soft agar assay, invasion assay, a xenograft mouse model, and a β-catenin-responsive SuperTopFlash luciferase assay.

Results: Compared with matched normal endometrial cases, Dkk3 was down-regulated in EC (p<0.0001). Among cancer cases, Dkk3 expression was significantly reduced in patients with higher stage (p=0.002), positive pelvic lymph nodes (p=0.0004), non-endometrioid histology (p=0.02), and cytology-positive ECs (p=0.02). Enforced expression of Dkk3 in EC cell lines showed reduced proliferation (p<0.0001), anchorage-independent growth (p=0.005), invasion (p=0.02), and reduced TCF activity (p=0.04), confirming Dkk3 as a negative regulator of the β-catenin/Wnt signaling pathway. Tumor growth in Dkk3-injected mice was not statistically different, though did plateau towards the end, and was associated with increased lymphoid infiltration and tumor necrosis.

Conclusion: Dkk3 gene expression is frequently downregulated in endometrial cancer, and is associated with poor prognostic clinicopathologic markers. The results also identify a role for Dkk3 as a tumor suppressor in EC, affecting both proliferation and invasiveness. These findings may prove to be important in the design of novel biomarkers and treatment modalities for advanced EC.

Fig. 1

Dkk3 expression is downregulated in endometrial cancer tissues. A, mRNA levels of Dkk3 in normal endometrial tissues and matched endometrial cancer tissues were determined by real-time RT-PCR. Percentage of loss of Dkk3 mRNA expression from normal endometrium to EC for each matched tissue pair is shown below the figure. B, mRNA levels in all normal endometrial tissues compared to all endometrial cancer tissues. C, Individual Dkk3 mRNA expression in all Stage I/II and Stage III/IV EC tumors. D, Dkk3 mRNA expression is reduced in Stage I/II compared to Stage III/IV EC tissues, as determined by real-time RT-PCR. All experiments were determined by real-time RT-PCR and performed in triplicates. * denotes p < 0.05, ** denotes p < 0.005, and *** denotes p < 0.0001.

Fig. 2

Dkk3 mRNA and protein expression are decreased in the endometrial cancer cell lines, ECC-1, HEC1A, and RL95-2, as compared to the benign endometrial cell line, HESC. A, mRNA levels of Dkk3 in HESC, ECC-1, HEC1A, and RL95-2, were determined by real-time RT-PCR. B, Expression of Dkk3 protein was determined by Western blotting with anti-Dkk3 antibodies in the same cell lines. All experiments were performed in triplicates. * denotes p < 0.05.

Fig. 3

Wnt3a activates β-catenin/TCF signaling in the endometrial cancer cell line ECC-1, but is inhibited by addition of exogenous or endogenous Dkk3. A, Addition of Wnt3a-conditioned medium results in a 15-fold increase in Wnt throughput in ECC-1, as determined by SuperTopFlash (STF) dual-luciferase assay. B, Cells endogenously producing Dkk3 following transient transfection exhibit a diminished response to Wnt3a. C, Exogenous Dkk3 CM reduces Wnt throughput in the presence and absence of Wnt3a. All experiments were performed in triplicates via SuperTopFlash dual-luciferase with Renilla normalization. * denotes p < 0.05.

Fig. 4

Dkk3 expression is decreased in the ECC-1 cell line, and Dkk3 transfection decreases proliferation, invasion and anchorage-independent growth in ECC-1 cells. A, mRNA levels of Dkk3 in stably-transfected ECC-1 cells (ECC1-Dkk3) were determined by real-time RT-PCR and compared to wild-type ECC-1 cells (wt-ECC1). B, Dkk3 reduces proliferation in ECC-1, as determined by cell proliferation assay. C, Dkk3 reduces anchorage-independent growth in ECC-1 cells, as determined by soft agar colony formation assay. D, Dkk3 decreases invasion in ECC-1 cells, as determined by Matrigel invasion assay. All experiments were performed in triplicate. * denotes p < 0.05, and *** denotes p < 0.0001.

Fig. 5

Xenograft mouse studies. A, Tumor volumes. Dkk3 expression in the xenograft mouse model results in no statistically significant difference in tumor volumes. B, Gross tumors showing necrosis and hemorrhage in Dkk3-expressing xenograft mice, compared to controls. C, Representative H&E stains of tumor from pCMV and Dkk3-expressing xenograft mice. i, Representative H&E stains of tumor from pCMV-ex show an endometrial adenocarcinoma with no necrosis and some glandular differentiation. ii, Representative H&E stains of tumor from Dkk3-expressing xenograft mice show an endometrial adenocarcinoma with large amounts of lymphoid infiltrate and necrosis. iii, Representative H&E stains of tumor from Dkk3-expressing xenograft mice show an adenocarcinoma with increased necrosis and hemorrhage.