FKBP3 aggravates the malignant phenotype of diffuse large B-cell lymphoma by PARK7-mediated activation of Wnt/β-catenin signalling

Abstract

Diffuse large B-cell lymphoma (DLBCL) is difficult to treat due to the high recurrence rate and therapy intolerance, so finding potential therapeutic targets for DLBCL is critical. FK506-binding protein 3 (FKBP3) contributes to the progression of various cancers and is highly expressed in DLBCL, but the role of FKBP3 in DLBCL and its mechanism are not clear. Our study demonstrated that FKBP3 aggravated the proliferation and stemness of DLBCL cells, and tumour growth in a xenograft mouse model. The interaction between FKBP3 and parkinsonism associated deglycase (PARK7) in DB cells was found using co-immunoprecipitation assay. Knockdown of FKBP3 enhanced the degradation of PARK7 through increasing its ubiquitination modification. Forkhead Box O3 (FOXO3) belongs to the forkhead family of transcription factors and inhibits DLBCL, but the underlying mechanism has not been reported. We found that FOXO3 bound the promoter of FKBP3 and then suppressed its transcription, eventually weakening DLBCL. Mechanically, FKBP3 activated Wnt/β-catenin signalling pathway mediated by PARK7. Together, FKBP3 increased PARK7 and then facilitated the malignant phenotype of DLBCL through activating Wnt/β-catenin pathway. These results indicated that FKBP3 might be a potential therapeutic target for the treatment of DLBCL.

Keywords: DLBCL; FKBP3; FOXO3; PARK7; lymphoma; wnt/β-catenin.

FIGURE 1

FKBP3 promoted the proliferation of DLBCL cells. (A) Real‐time PCR and western blot analysis were used to detect the mRNA and protein expression of FKBP3 in DB cells and farage cells. (B) Cell viability analysis was performed by CCK‐8 after transfection 0, 24, 48 and 72 h. The optical density value meant the cell viability. (C) The protein expression of PCNA in DB cells and farage cells was detected by western blot analysis. (D) EdU in DB cells and farage cells measured by a fluorescence microscope IX53. EdU‐incorporated signal was red and nucleus was stained blue with DAPI in DB cells and farage cells. Bars, 50 μm, ×400. Data were expressed as mean ± SD (n = 3). ^# p < 0.05 compared to vector 1 or siNC. ^## p < 0.01 compared to vector 1 or siNC. **P < 0.01 compared to siNC. CCK‐8, cell counting kit‐8; DAPI, 4′,6‐diamidino‐2‐phenylindole; DLBCL, diffuse large B‐cell lymphoma; EdU, 5‐ethynyl‐2′‐deoxyuridine; FKBP3, FK506 binding protein 3; PCNA, proliferating cell nuclear antigen; PCR, polymerase chain reaction; SD, standard deviation.

FIGURE 2

Knockdown of FKBP3 induced G1 arrest of DLBCL cells. (A) The effect of FKBP3 overexpression on cell cycle was detected by a flow cytometer NovoCyte. The PI was used to dye. (B) Cell cycle was analysed using flow cytometry in DB and farage cells with silenced FKBP3. The PI was used to dye. (C) The protein of cyclin D1, cyclin B1, CDK4 and CDK1 was measured by western blot analysis. Data were expressed as mean ± SD (n = 3). ^# p < 0.05 compared to vector 1 or siNC. ^## p < 0.01 compared to vector 1 or siNC. CDK4, cyclin dependent kinase 4; DLBCL, diffuse large B‐cell lymphoma; FKBP3, FK506 binding protein 3; PI, propidium iodide; SD, standard deviation.

FIGURE 3

FKBP3 stimulated the expression of stemness markers in DLBCL cells. The mRNA of FKBP3 (A), Nestin (B), CD133 (C), OCT4 (D) and SOX2 (E) in DB cells and farage cells was analysed by real‐time PCR. (F) The CD133 (red) protein expression was measured by immunofluorescence. Nucleus was stained blue with DAPI in DB cells and farage cells. Bars, 50 μm, ×400. Data were expressed as mean ± SD (n = 3). ^## p < 0.01 compared to vector 1 or siNC. CD133, prominin 1; DAPI, 4′,6‐diamidino‐2‐phenylindole; DLBCL, diffuse large B‐cell lymphoma; FKBP3, FK506 binding protein 3; OCT4, POU class 5 homeobox 1; PCR, polymerase chain reaction; SD, standard deviation; SOX2, SRY‐Box transcription factor 2.

FIGURE 4

FKBP3 boosted tumour growth of mice with DLBCL xenograft. (A) Tumour volume on day 4, 8, 12, 16 and 21, and tumour images on day 21 in each group were shown. (B) Real‐time PCR was used to detect the mRNA expression of FKBP3 in tumour. (C) IHC was used to detect the PCNA expression in tumour. Lower panels showed the magnified images of regions framed by red rectangles on the upper panels. Bars, 200 or 50 μm, ×100 or ×400. (D) The mRNA expression of OCT4 and SOX2 was measured by real‐time PCR. Data were expressed as mean ± SD (n = 6). ^## p < 0.01 compared to vector 1 or shNC. **p < 0.01 compared to shNC. DLBCL, diffuse large B‐cell lymphoma; FKBP3, FK506 binding protein 3; IHC, immunohistochemistry; OCT4, POU class 5 homeobox 1; PCNA, proliferating cell nuclear antigen; PCR, polymerase chain reaction; SD, standard deviation; SOX2, SRY‐Box transcription factor 2.

FIGURE 5

FKBP3 promoted the stability of PARK7. (A) PARK7 expression in DLBCL and paired normal sample in the GEPIA database. The correlation analysis of PARK7 and FKBP3. (B) Western blot analysis was used to detect the protein expression of FKBP3 and PARK7 in DB cells and farage cells. (C) The expression and localization of FKBP3 (red) and PARK7 (green) in DB cells and farage cells were determined by immunofluorescence assay. The nucleus was stained blue with DAPI. Bars, 50 μm, ×400. (D) CoIP was used to detect the interaction between FKBP3 and PARK7 in DB cells. (E) The protein expression of PARK7 in DB cells after treatment with CHX or CHX + MG132 for 0, 1, 3, 6 and 9 h was measured by western blot analysis. (F) The binding of PARK7 and Ubi was analysed by CoIP in DB cells. Data were expressed as mean ± SD (n = 3). *P < 0.05 compared to normal samples. CHX, cycloheximide; CoIP, co‐immunoprecipitation; DAPI, 4′,6‐diamidino‐2‐phenylindole; FKBP3, FK506 binding protein 3; PARK7, parkinsonism associated deglycase; SD, standard deviation; Ubi, ubiquitin.

FIGURE 6

FKBP3 activated Wnt/β‐catenin signalling pathway and aggravated the malignant phenotype of DLBCL cells through increasing PARK7 expression. (A) The protein expression of PARK7 and active β‐catenin was measured by western blot analysis. (B) Real‐time PCR was used to detect the mRNA expression of cyclin D1 and MYC in DB cells. (C) Cell viability of DB cells was analysed by CCK‐8. The optical density value meant the cell viability. (D) The mRNA expression of Nestin, CD133, OCT4 and SOX2 was measured by Real‐time PCR. (E) Western blot analysis was used to detect the protein expression of FKBP3, active β‐catenin in DB cells. (F) The mRNA expression of cyclin D1 and MYC in DB cells was analysed by real‐time PCR. (G) The transcriptional activity of β‐catenin was detected by luciferase reporter assay. (H) The protein expression of PARK7 and active β‐catenin was measured by western blot analysis. (I) Real‐time PCR was used to detect the mRNA expression of cyclin D1 and MYC in DB cells. (J) Cell viability of DB cells was analysed by CCK‐8. The optical density value meant the cell viability. (K) The mRNA expression of PCNA, OCT4 and SOX2 was measured by Real‐time PCR. Data were expressed as mean ± SD (n = 3). ^# p < 0.05 compared to vector 2. *p < 0.05 compared to normal or oeFKBP3 + siNC. ^## p < 0.01 compared to vector 1, vector 2 or TOP +vector 1. **p < 0.01 compared to oeFKBP3 + siNC. CCK‐8, cell counting kit‐8; CD133, prominin 1; DLBCL, diffuse large B‐cell lymphoma; FKBP3, FK506 binding protein 3; MYC, MYC Proto‐Oncogene, BHLH Transcription Factor; OCT4, POU class 5 homeobox 1; PARK7, parkinsonism associated deglycase; PCNA, proliferating cell nuclear antigen; PCR, polymerase chain reaction; SD, standard deviation; SOX2, SRY‐Box transcription factor 2.

FIGURE 7

FKBP3 transcription was suppressed by FOXO3. (A) Real‐time PCR and (B) western blot analyses were used to detect the mRNA and protein expression of FOXO3 and FKBP3 in DLBCL cells. (C) FKBP3 transcription in oeFOXO3‐transfected DB cells was measured by luciferase reporter assay. The region of FKBP3 promoter was cloned into the pGL3 reporter vector. (D) ChIP was performed to detect the binding of FOXO3 to FKBP3 promoter in DB cells. Data were expressed as mean ± SD (n = 3). ^## p < 0.01 compared to vector 4 or vector 4 + pGL3 promoter (−995/+19, −634/+19, −521/+19). ChIP, chromatin immunoprecipitation; DLBCL, diffuse large B‐cell lymphoma; FKBP3, FK506 binding protein 3; FOXO3, Forkhead Box O3; PCR, polymerase chain reaction; SD, standard deviation.