The F-box E3 ligase protein FBXO11 regulates EBNA3C-associated degradation of BCL6

Abstract

Most mature B-cell malignancies originate from the malignant transformation of germinal center (GC) B cells. The GC reaction appears to have a role in malignant transformation, in which a major player of the GC reaction is BCL6, a key regulator of this process. We now demonstrate that BCL6 protein levels were dramatically decreased in Epstein-Barr virus (EBV)-positive lymphoblastoid cell lines and Burkitt's lymphoma cell lines. Notably, BCL6 degradation was significantly enhanced in the presence of both EBNA3C and FBXO11. Furthermore, the amino-terminal domain of EBNA3C, which contains residues 50-100, interacts directly with FBXO11. The expression of EBNA3C and FBXO11 resulted in a significant induction of cell proliferation. Furthermore, BCL6 protein expression levels were regulated by EBNA3C via the Skp Cullin Fbox (SCF)^FBXO11 complex, which mediated its ubiquitylation, and knockdown of FBXO11 suppressed the transformation of lymphoblastoid cell lines. These data provide new insights into the function of EBNA3C in B-cell transformation during GC reaction and raise the possibility of developing new targeted therapies against EBV-associated cancers.

Importance: The novel revelation in our study involves the suppression of BCL6 expression by the essential Epstein-Barr virus (EBV) antigen EBNA3C, shedding new light on our current comprehension of how EBV contributes to lymphomagenesis by impeding the germinal center reaction. It is crucial to note that while several EBV latent proteins are expressed in infected cells, the collaborative mechanisms among these proteins in regulating B-cell development or inducing B-cell lymphoma require additional investigation. Nonetheless, our findings carry significance for the development of emerging strategies aimed at addressing EBV-associated cancers.

Keywords: E3 ligase; EBNA3C; Epstein-Barr virus; FBXO11; ubiquitin.

Fig 1

BCL6 level is reduced in latency III EBV-positive cells. (A) Fifteen million BJAB, LCL1, Mutu I, Sav I, and Kem I cells and corresponding latency III EBV-positive cells and (B) eight latent genes (EBNAs 1, 2, 3A, 3B, 3C, EBNA-LP, LMP1, and LMP2A) expressing stable BJAB cells were harvested and lysed with RIPA buffer. The expression levels of E3C, FBXO11, and BCL6 were detected by western blot. The relative density (RD) of BCL6 was quantitated for a representive blot and shown.

Fig 2

BCL6 levels are decreased in the presence of EBNA3C. Half a million Saos-2 cells were transfected with HA-BCL6 and (A) an increasing amount of Myc-tagged EBNA3C (10 and 15 µg) alone or together with Flag-FBXO11, (B) Myc-tagged EBNA3C (15 µg), Flag-FBXO11, and Flag-FBXO11mut separately or together. Total amounts of plasmids were kept constant by co-transfecting with the vector. At 48 hours post-transfection, the cells were harvested and lysed with RIPA buffer. The expression levels of E3C, FBXO11, and BCL6 were detected by western blot. The relative density (RD) of BCL6 was quantitated for a representative blot and shown.

Fig 3

EBNA3C associates with FBXO11 in molecular complexes in human cell lines. (A) Ten million Saos-2 cells were transfected with Flag-tagged FBXO11 and Flag-tagged FBXO11mut alone or together with Myc-tagged EBNA3C. At 48 hours post-transfection, the cells were harvested and lysed for immunoprecipitation with 2 µg anti-Flag antibody. The input and immunoprecipitated samples fractionated, and specific signals were detected by western blot by using antibodies against Flag and Myc. (B) EBNA3C associated with endogenous FBXO11. Sixty million BJAB, BJAB7, and LCL1 were collected and lysed for immunoprecipitation with 2 µg anti-FBXO11 antibody. Western blot was used to detect specific signals in the inputs and immunoprecipitated samples. (C) The schematic diagram summarizes the binding domains between different regions of EBNA3C. Jκ, RBP-Jκ; LZ, leucine zipper domain; AD, acidic domains; P rich, proline-rich; Q rich, glutamine-proline-rich; and NLS, nuclear localization signal. (D and E) The N terminus of EBNA3C is critical for EBNA3C and FBXO11 interaction. Ten million Saos-2 cells were transfected with Flag-tagged FBXO11 alone or together with Myc-tagged full-length EBNA3C or EBNA3C truncated mutants. At 48 hours post-transfection, the cells were harvested and lysed for immunoprecipitation with 2 µg anti-Flag antibody. The input and immunoprecipitated samples were resolved using 8% polyacrylamide (D) and 12% polyacrylamide (E) and blotted using specific antibodies.

Fig 4

Co-localization of EBNA3C and FBXO11 in human cells. (A) EBNA3C co-localized with FBXO11 in 293T and Saos-2 cells. A total of 0.1 million Saos-2 cells were plated on coverslips and transfected with Myc-tagged EBNA3C and Flag-tagged FBXO11. At 24 hours post-transfection, cells were subjected to immunofluorescence assays. (B) EBNA3C co-localized with endogenous FBXO11 in B cells. BJAB, BJAB7, and LCL1 cells were plated on the slide and air-dried. The cells were fixed and subjected to immunofluorescence assays as described in Materials and Methods. The colocalization between the green and red channels was calculated using Pearson’s coefficient (r) in ImageJ software (17).

Fig 5

EBNA3C and FBXO11 promote cell proliferation. (A and B) Saos-2 cells were transfected with the indicated plasmids and eGFP. The cells were selected with G418 (neomycin) antibiotics for 2 weeks. The cells were fixed, and the cell colonies were stained with 0.1% crystal violet. The relative colony number was measured by Image J software. (C) 5 × 10⁵ selected cells were plated and cultured for 6 days. Viable cells were counted every day using trypan blue staining.

Fig 6

EBNA3C mediates BCL6 degradation through the ubiquitin pathway. EBNA3C enhanced poly-ubiquitination of BCL2. Ten million Saos-2 cells were transfected with the indicated constructs. At 24 hours post-transfection, cells were incubated with 10 µM MG132 for another 16 hours. Then, the cells were harvested and subjected to immunoprecipitation using an antibody against BCL6. The input and immunoprecipitated samples were detected by western blot.

Fig 7

Knockdown of FBXO11 suppresses the transformation activity of LCL1. (A)Saos-2 and HEK293 cells were transfected with Flag-tagged FBXO11 and sh-Ctrl, or sh-FBXO11. After 24 hours, the cells were harvested, and the expression of FBXO11 was detected by western blot. (B) FBXO11 knockdown (shFBXO11) or control (shCtrl) stable BJAB or LCL1 cells were harvested, and FBXO11 mRNA expression was detected using real-time PCR. (C) FBXO11 knocked down BJAB and LCL1 cells were constructed by lentivirus transduction and selected by puromycin for 3 weeks. GFP fluorescence was determined in the selected cells. (D and E) Colony formation was measured in FBXO11 knocked down BJAB and LCL1 cells by soft agar assays. The relative colony number was measured by Image J software. (F and G) FBXO11 knocked down BJAB and LCL1 cells were stained with PI staining buffer and analyzed by flow cytometry.

Fig 8

Schematic diagram illustrating how EBNA3C promotes the degradation of FBXO11. In normal cells, the levels of FBXO11 were maintained to a minimal level that led to the restoration of BCL6 levels within the cell. In DLBCL, FBXO11 interacts with EBNA3C and acts as E3 ubiquitin ligase to polyubiquitinylate and degrades BCL6.