Fibulin-4 is associated with prognosis of endometrial cancer patients and inhibits cancer cell invasion and metastasis via Wnt/β-catenin signaling pathway

Abstract

Fibulin-4, an extracellular glycoprotein, which plays significant roles in elastic fiber assembly, is correlated to the progression of some cancers. However, the role of fibulin-4 in endometrial cancer cell invasion and metastasis remains unexplored. In our study, fibulin-4 expression was assessed by immunohistochemistry (IHC) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in normal endometrial tissues and endometrial carcinoma tissues. Using single cell cloning, strongly, and weakly, invasive subclones were derived from KLE and Ishikawa endometrial carcinoma cell lines. RT-qPCR, western blotting, and immunocytochemistry (ICC) were used to assess mRNA and protein expressions of fibulin-4 in primary cultured endometrial cells, 4 types of endometrial cancer cell lines, and the different invasive subclones. Using lentivirus transfection, fibulin-4 shRNA and pLVX-fibulin-4 were constructed and used to infect the strongly and weakly invasive subclones. The effects of fibulin-4 on the biological characteristics of endometrial carcinoma cells were detected by cell functional assays in vitro and in vivo. Using Wnt signaling pathway inhibitor XAV-939 and activator LiCl, we detected the role of fibulin-4 in the Wnt/β-catenin pathway and the relationship with epithelial to mesenchymal transition (EMT). Fibulin-4 was decreased in endometrial carcinoma tissues, and loss of fibulin-4 expression was significantly related with poor differentiation, lymph node metastasis, and poor prognosis of endometrial carcinoma. Fibulin-4 significantly inhibited endometrial carcinoma cell proliferation, invasion, metastasis, and EMT through the Wnt/β-catenin pathway. Fibulin-4 has the ability to suppress endometrial cancer progression. These results can contribute to the development of a new potential therapeutic target for patients with endometrial carcinoma.

Keywords: endometrial cancer; epithelial-mesenchymal transition (EMT); fibulin-4; invasion; metastasis.

Figure 1. Expressions of fibulin-4 in human endometrial tissues

The proliferative stage of normal human endometrium (A), the secretory phase of normal human endometrium (B), human endometrium with complex hyperplasia (C), particular atypical endometrial hyperplasia (D), well differentiation of endometrial carcinoma (E), poor differentiation of endometrial carcinoma (F), the area of squamous differentiation (G), the clear cell carcinoma (H) and the serous carcinoma (I) were measured by IHC. (Magnification×200). (J) Western blot analysis was performed to detect fibulin-4 in a set to paired normal-tumor tissue samples. (K) Kaplan-Meier curve showing overall survival. (L) Kaplan-Meier curve showing progression-free survival. Patients with low fibulin-4 expression (green line, n = 171) had much worse prognosis than those with high fibulin-4 expression (blue line, n = 29).

Figure 2. Different proliferation, migration and invasion abilities of 4 types of human endometrial cancer cell lines

(A) The growth curves of human endometrial cancer cells showed that KLE and HEC-1A cells had higher proliferation abilities compared to Ishikawa and HEC-1B cells. (B) The colony numbers formed by KLE and HEC-1A cells were significantly higher than those formed by Ishikawa and HEC-1B cells. (C) The colony images of human endometrial cancer cells as examined by soft agar colony formation assay. (D) The images of cells migrating PVPF filters as examined by cell migration assay using Boyden chambers. (E) The images of cells invading Matrigel-coated membranes as examined by cell invasion assay using Boyden chambers. (F) The average migrating cell counts of KLE and HEC-1A cells were much higher than those of Ishikawa and HEC-1B cells. (G) The average invading cell counts of KLE and HEC-1A cells were much higher than those of Ishikawa and HEC-1B cells. (Magnification ×200).*P < 0.05 versus control.

Figure 3. Fibulin-4 expressions in endometrial carcinoma cells and normal endometrial cell

Fibulin-4 expressions of KLE, Ishikawa, HEC-1A, HEC-1B and normal endometrial cells as measured by (A) ICC staining (Magnification×200), (B) western blot and (C) q-RT-PCR. The strongest expression of fibulin-4 was detected in normal endometrial cells, and compared to Ishikawa and HEC-1B cells, fibulin-4 was weakly expressed in KLE and HEC-1A cells, which had higher proliferation and invasion abilities. *P < 0.05 versus control.

Figure 4. Fibulin-4 expressions in strongly invasive subclones and weakly invasive subclones

Fibulin-4 expressions in strongly invasive subclones and weakly invasive subclones as measured by (A) ICC staining (Magnification×200), (B) q-RT-PCR and (C) western blot. In contrast with the weakly invasive subclones, low fibulin-4 expression was found in strongly invasive subclones KLE-1 and ISK-1.*P < 0.05 versus control.

Figure 5. Identification of the transfection efficiencies after lentivirus-mediated transfection

Fibulin-4 expressions in lentivirus-infected cells as measured by (A) western blot, (B) ICC staining (Magnification ×200), and (C) q-RT-PCR. After viral infection, fibulin-4 expression was decreased in the weakly invasive subclones KLE-28 and ISK-23, meanwhile, fibulin-4 expression was increased in the strongly invasive subclones KLE-1 and ISK-1.*P < 0.05 versus control.

Figure 6. Effects of fibulin-4 on cell proliferation and colony formation abilities

(A) Knockdown of fibulin-4 promoted cell proliferation abilities of the weakly invasive subclones, meanwhile, over-expression of fibulin-4 inhibited cell proliferation abilities of the strongly invasive subclones. (B) Fibulin-4 knockdown increased the colony forming capacities of weakly invasive subclone, while over-expression of fibulin-4 decreased the colony forming capacities of strongly invasive subclone. *P < 0.05 versus control.

Figure 7. Effects of fibulin-4 on cell migration abilities

(A) Cell migration images of lentivirus-infected cells as measured by Boyden chambers without Matrigel. (Magnification ×200). (B) Fibulin-4 knockdown promoted cell migrating abilities, while fibulin-4 over-expression suppressed cell migrating abilities. *P < 0.05 versus control.

Figure 8. Effects of fibulin-4 on cell invasion abilities

(A) Cell invasion images of lentivirus-infected cells by Boyden chambers coated with Matrigel. (Magnification ×200). (B) Fibulin-4 knockdown promoted cell invading abilities, while fibulin-4 over-expression suppressed cell invading abilities. *P < 0.05 versus control.

Figure 9. Effects of fibulin-4 on tumor growth and lung metastasis in nude mice

(A) Tumor growths of lentivirus-infected cells were observed continuously for 8 weeks. The average tumor volumes of fibulin-4 shRNA infected KLE-28 cells and negative control KLE-1 cells were much higher than those formed by negative control KLE-28 cells and pLVX-fibulin-4-infected KLE-1 cells. (B) Photograph of xenografts dissected from nude mice after subcutaneous inoculation. (C) H&E staining were performed on lung metastasis after inoculation through tail vein. The pLVX-fibulin-4-infected group and the weakly invasive subclone group had no lung metastasis; however, the lung metastasis rates of the fibulin-4 knockdown group and the strongly invasive group were 80% and 100%, respectively. (Magnification ×200). *P < 0.05 versus control.

Figure 10. Effects of fibulin-4 knockdown on EMT genes correlated to tumor progression

After RNA interference, EMT markers, including E-cadherin, N-cadherin, vimentin, Snail Slug and Twist as measured by (A) Western blot and (B) real-time qPCR in the fibulin-4 shRNA infected groups. The results showed that fibulin-4 knockdown significantly decreased the expression of E-cadherin, and increased the expression of N-cadherin, vimentin, snail, slug and twist. *P < 0.05 versus control.

Figure 11. Effects of fibulin-4 over-expression on EMT genes correlated to tumor progression

After transfection, EMT markers, including E-cadherin, N-cadherin, vimentin, Snail Slug and Twist as measured by (A) Western blot and (B) real-time qPCR in the pLVX-fibulin-4 infected groups. The results showed that fibulin-4 up-regulation could increase the expression of E-cadherin, and decrease the expression of N-cadherin, vimentin, snail, slug and twist. (C) After co-culturing normal endometrial fibroblasts and pLVX-fibulin-4-infected KLE-1 for 24 h, 48 h, and72 h, the fibroblasts markers vimentin and alpha-smooth muscle actin (α-SMA) were significantly decreased with the extension of co-culture time. *P < 0.05 versus control.

Figure 12. Effects of fibulin-4 on the Wnt/β-catenin and Notch pathways

(A) Fibulin-4 knockdown increased the expression of β-catenin, C-myc, and cyclin D1, activating the Wnt/β-catenin pathway, but had no obvious effects on the Notch pathway. (B) Fibulin-4 upregulation decreased the expression of β-catenin, C-myc, and cyclin D1, deactivated the Wnt/β-catenin pathway, but had no obvious effects on the Notch pathway.

Figure 13. Inhibition and promotion the Wnt/β-catenin pathway

(A) In the fibulin-4 shRNA-infected cells, Wnt signaling pathway inhibitor XAV-939 could significantly inhibit the Wnt/β-catenin pathway and EMT, both of which were activated by fibulin-4 knockdown. (B) In the pLVX-fibulin-4-infected cells, Wnt signaling pathway activator LiCl could promote the Wnt/β-catenin pathway and EMT, both of which were deactivated by fibulin-4 upregulation.