FBXL16 Promotes Endometrial Progesterone Resistance via PP2AB55 α /Cyclin D1 Axis in Ishikawa

Abstract

Background: F-box proteins are essential components of the E3 ubiquitin ligases which are involved in the regulation of almost all life activities such as cell cycle, proliferation, and apoptosis, which have become an important research and drug target. However, there are few studies on F-box and leucine-rich repeat protein 16 (FBXL16) in endometrial carcinoma.

Methods: Clinical samples were collected for determining the correlation between FBXL16 and endometrial carcinoma. Cells were screened and established with Ishikawa cells which proved the fundamental role of FBXL16 in regulating cell proliferation and cell cycle. The MPA-resistant endometrial carcinoma cell line Ishikawa/MPA was established. FBXL16, PP2A^B55α , and cyclin D1 were analyzed separately in MPA sensitive and resistant Ishikawa cells in vitro and in vivo.

Results: The high expression of FBXL16 was positively correlated with MPA resistance and poor prognosis of endometrial cancer. MPA tolerance of endometrial cancer cells was inhibited by knockdown of FBXL16 in DNA content assessment, CCK-8, and colony formation. It was confirmed that FBXL16 inhibited the activity of substrate PP2A^B55α by binding to PP2A, reduced the phosphorylation level at Thr308 site of AKT1, inhibited the expression of GSK-3β, and thus led to a significant decrease in the phosphorylation level of cyclin D1, which prevented the ubiquitination recognition and degradation of cyclin D1.

Conclusion: In our experiments, FBXL16 binds PP2A to promote the dephosphorylation of Thr286 site of cyclin D1 via AKT1/GSK3β/cyclin D1 pathway, which is required for resisting the ubiquitination degradation and enhances the MPA resistance of Ishikawa.

Figure 1

FBXL16 is highly expressed in MPA-resistant endometrial cancer tumors. (a) Box plot from GEPIA2 database indicated the expression of FBXL16 in UCEC tissues. (b) RT-qPCR examined the expression of FBXL16 in endometrial cancer tissues and paracancer tissues (group = 2, n = 30). (c) Immunohistochemistry analysis of FBXL16 expression in endometrial cancer tissues and paracancer tissues. (d) RT-qPCR assessed the expression of FBXL16 in EC/r and EC/s (group = 2, n = 15). Results are presented as mean ± SD. ^∗P < 0.05. ^∗∗P < 0.01. ^∗∗∗P < 0.001.

Figure 2

FBXL16 knockdown inhibited cell proliferation and MPA resistance of Ishikawa. (a) RT-qPCR examined the expression of FBXL16 in endometrial cancer cell lines in comparison with human immortalized endometrial epithelial cells (EEC-1). (b) RNA level of FBXL16 validated by RT-qPCR in parent Ishikawa cells (Ishikawa/P) and Ishikawa cells resistant to MPA (Ishikawa/MPA). (c) Transfection efficiency of shFBXL16 in transfected Ishikawa/MPA cells by RT-qPCR. (d) Colony formation assay of Ishikawa/MPA with the shRNA-NC group or shFBXL16 group. (e) EdU incorporation assay of Ishikawa/MPA with the shRNA-NC group or shFBXL16 group. (f) Apoptosis detection of Ishikawa/MPA with the shRNA-NC group or shFBXL16 group by Annexin V-APC/7-AAD method. (g) DNA content detection of Ishikawa/MPA with the shRNA-NC group or shFBXL16 group by PI staining. Results are presented as mean ± SD. ^∗P < 0.05. ^∗∗P < 0.01. ^∗∗∗P < 0.001.

Figure 3

FBXL16 stabilized cyclin D1 protein by indirect interactions. (a, b) Ishikawa/MPA cells with shRNA-NC or shFBXL16 were pretreated with CHX for 90 min, and protein expression of cyclin D1 and cyclin E was analyzed by western blot at indicated times (left) and quantitatively analyzed (right). (c) Coimmunoprecipitation of FBXL16 and cyclin D1 in Ishikawa/MPA cells with the shRNA-NC or shFBXL16 group. Results are presented as mean ± SD. ^∗P < 0.05. ^∗∗P < 0.01. ^∗∗∗P < 0.001.

Figure 4

FBXL16 dephosphorylated cyclin D1 by regulating Akt1/GSK-3β signaling pathway via binding PP2A^B55α. (a) Western blot analyses of cyclin D1 and phosphor-cyclin D1 (T286) in Ishikawa/MPA with shNC and shFBXL16 cells. (b) Coimmunoprecipitation of E3 ligase and cyclin D1 in Ishikawa/MPA cells with the shRNA-NC or shFBXL16 group. (c) Coimmunoprecipitation of FBXL16, PP2A-A α/β, PP2A-B55α, and PP2A-C in Ishikawa/MPA cells with the shRNA-NC or shFBXL16 group. (d) Western blot analyses of phosphor-cyclin D1 (T286) and GSK3β/AKT1 pathway in Ishikawa/MPA with shNC and shFBXL16 cells. Results are presented as mean ± SD. ^∗P < 0.05. ^∗∗P < 0.01. ^∗∗∗P < 0.001.

Figure 5

FBXL16 promotes MPA resistance and proliferation of endometrial cancer tumors in vivo. (a) The xenografts from nude mice inoculated in Ishikawa-MPA/shNC and Ishikawa-MPA/shFBXL16 cells. The tumor volumes in nude mice and mouse weight were determined for 27 days. Mean tumor weights at day 27 were determined. (b) H-E staining and IHC analysis of Ki-67, FBXL16, and phosphor-cyclin D1 (T268) were performed in the tumor tissue sections of Ishikawa-MPA/shNC and Ishikawa-MPA/shFBXL16. Data are represented as mean ± SD (n = 5 per group).