FKBP14 overexpression contributes to osteosarcoma carcinogenesis and indicates poor survival outcome

Abstract

The FK506-binding protein 14 (FKBP14) is a subfamily of immunophilins, has been implicated in various biochemical processes. However, its effects on the primary malignant bone tumor, osteosarcoma, are unclear. Here, we reported that FKBP14 may be an oncogene as it overexpressed in osteosarcoma tissues and cell lines, and FKBP14 expression was correlated with metastases, recurrence, tumor maximum diameter and poor survival time. FKBP14 was associated with the biological pathways including cell cycle, apoptosis and metastasis. Furthermore, we detected FKBP14 knockdown induced cell cycle arrest, apoptosis, invasion and adhesion in vitro. FKBP14 knockdown decreased the protein levels of PCNA, CDK1 and CCNB1 that promotes cell cycle, increased Bax, caspase-3 and caspase-7 protein involved in promoting cell apoptosis, and increased KIF4A expression as well as decreased SMC4 and TMEM33 proteins that contribute to cell invasion and adhesion. In addition, FKBP14 knockdown also caused a significant inhibition in tumor growth in vivo. Then, we found that the protein RhoA was identified as a binding partner of FKBP14. Taken together, FKBP14 may act as an oncogene in osteosarcoma via suppressing apoptosis and promoting invasion and adhesion in osteosarcoma carcinogenesis. FKBP14 may be a prognostic factor and potential target for osteosarcoma treatment.

Keywords: FKBP14; GSEA; apoptosis; osteosarcoma; proliferation.

Figure 1. FKBP14 up-regulation in osteosarcoma tissues and regulates biological pathways

(A) Microarray analysis of FKBP14 expression in bone cysts and osteosarcoma in Xiaoshan Chinese Medical Hospital; (B) Representative immunohistochemical staining for FKBP14 in osteosarcoma tissues. Survival analysis showed that FKBP14-lower expression tumors have a favorable prognosis compared with FKBP14-higher expression tumors; (C) The overall survival time of 150 osteosarcoma patients from Xiaoshan Chinese Medical Hospital; (D) The metastase-free survival time of 81 osteosarcoma patients from Xiaoshan Chinese Medical Hospital; (E) The overall survival time of 37 osteosarcoma patients from GSE39055 database. GSEA compared FKBP14 higher expression group (red) against FKBP14 lower expression group (blue) of osteosarcoma patients in the E-MEXP-3628 dataset. Enrichment plots are shown for a set of activated genes related to (F) cell cycle, (G) apoptosis and (H) metastasis. **P < 0.01 compared with bone cysts.

Figure 2. FKBP14 expression in osteosarcoma cell lines

FKBP14 expression was measured by (A) Real-time PCR and (B) Western blot; Knockdown of FKBP14 by shRNA showed notably expression inhibited in (C) mRNA and (D) protein levels in U-2OS and MG63 cells; Overexpression of FKBP14 showed notably expression increased in (E) mRNA and (F) protein levels in 143B cells. **P < 0.01 compared with NC group. NC: negative control.

Figure 3. FKBP14 knockdown inhibits cell proliferation in vitro and in vivo

(A, B) FKBP14 knockdown inhibits U-2OS and MG63 cells proliferation in vitro; (C) FKBP14 overexpression promotes 143B cells proliferation in vitro; (D) FKBP14 expression was measured by Western blot; (E) FKBP14 knockdown inhibits tumor growth in nude mice xenograft model in vivo; (F) Tumor volume and (G) weight were also measured after FKBP14 knockdown. **P < 0.01 compared with NC group. NC: negative control.

Figure 4. FKBP14 knockdown induces cell cycle arrest

Cell cycle arrested at G1 phase after FKBP14 knockdown in (A) U-2OS and (B) MG63 cells assessed by flow cytometry; Expressions of cell cycle markers (PCNA, CDK1 and CCNB1) were significantly down-regulated after FKBP14 knockdown in (C) U-2OS and (D) MG63 cells. **P < 0.01 compared with NC group. NC: negative control.

Figure 5. FKBP14 knockdown induces cell apoptosis

Cell apoptosis was significantly increased after FKBP14 knockdown in (A) U-2OS and (B) MG63 cells and decreased in (C) 143B cells with FKBP14 overexpression, assessed by flow cytometry; Expressions of apoptosis markers (Bax, caspase-3 and caspase-7) were significantly up-regulated after FKBP14 knockdown in (D) U-2OS and (E) MG63 cells and were down-regulated after FKBP14 overexpression in (F) 143B cells. **P < 0.01 compared with NC group. NC: negative control.

Figure 6. FKBP14 knockdown inhibits cell invasion and adhesion

(A) Cell invasion and (B) adhesion were significantly inhibited after FKBP14 knockdown in U-2OS and MG63 cells and were increased in 143B cells, respectively. **P < 0.01 compared with NC group. NC: negative control.

Figure 7. Effect of FKBP14 knockdown on the expression of proteins correlated with invasion and adhesion

Expressions of invasion and adhesion markers were significantly up-regulated (KIF4A) or down-regulated (SMC4 and TMEM33) after FKBP14 knockdown in (A) U-2OS and (B) MG63 cells; (C) Expressions of other invasion and adhesion markers were significantly down-regulated (E-cadherin) or up-regulated (Twist1, Snail1, Snail2, MMP2 and MMP9) after FKBP14 overexpression in 143B cells. **P < 0.01 compared with NC group. NC: negative control.

Figure 8. FKBP14 binds to RhoA in vitro

Co-immunoprecipitation showed that FKBP14 interacts with RhoA in the cell lines (A) U-2OS and (B) MG63 cells.