BCLAF1 binds SPOP to stabilize PD-L1 and promotes the development and immune escape of hepatocellular carcinoma

Interaction between programmed death-1 (PD-1) ligand 1 (PD-L1) on tumor cells and PD-1 on T cells allows tumor cells to evade T cell-mediated immune surveillance. Strategies targeting PD-1/PD-L1 have shown clinical benefits in a variety of cancers. However, limited response rates in hepatocellular carcinoma (HCC) have prompted us to investigate the molecular regulation of PD-L1. Here, we identify B cell lymphoma-2-associated transcription factor 1 (BCLAF1) as a key PD-L1 regulator in HCC. Specifically, BCLAF1 interacts with SPOP, an E3 ligase that mediates the ubiquitination and degradation of PD-L1, thereby competitively inhibiting SPOP-PD-L1 interaction and subsequent ubiquitination and degradation of PD-L1. Furthermore, we determined an SPOP-binding consensus (SBC) motif mediating the BCLAF1-SPOP interaction on BCLAF1 protein and mutation of BCLAF1-SBC motif disrupts the regulation of the SPOP-PD-L1 axis. In addition, BCLAF1 expression was positively correlated with PD-L1 expression and negatively correlated with biomarkers of T cell activation, including CD3 and CD8, as well as with the level of immune cell infiltration in HCC tissues. Besides, BCLAF1 depletion leads to a significant reduction of PD-L1 expression in vitro, and this reduction of PD-L1 promoted T cell-mediated cytotoxicity. Notably, overexpression of BCLAF1 sensitized tumor cells to checkpoint therapy in an in vitro HCC cells-Jurkat cells co-culture model, whereas BCLAF1-SBC mutant decreased tumor cell sensitivity to checkpoint therapy, suggesting that BCLAF1 and its SBC motif serve as a novel therapeutic target for enhancing anti-tumor immunity in HCC. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-024-05144-z.

Western blot of the products of in vivo ubiquitination assays performed using WCLs from HEK-293T cells transfected with the indicated plasmids.f qRT-PCR measurement of SPOP mRNA levels in BCLAF1-depleted and BCLAF1-overexpressed SK-Hep1 cells.g Western blot of WCLs from BCLAF1-depleted and BCLAF1-overexpressed SK-Hep1 cells.h SK-Hep1 cells were treated with MG-132 (20 μM) for 8 h prior to lysis, and in vivo ubiquitination assays were performed using the antibodies shown to detect the effect of BCLAF1 on ubiquitination of endogenous PD-L1 mediated by SPOP.
i Western blot of ubiquitination levels of PD-L1 in HCC tissues.j HepG2 and SK-Hep1 cells were infected with lentiviruses expressing SPOP-specific sh RNAs (sh SPOP#1, #2, #3), SPOP-specific overexpression (SPOP-OE), or a control (Control) of the same vector system for 72 h.After infection, WCLs were prepared and SPOP protein levels were determined by Western blot.All quantitation were normalized to the protein levels of GAPDH in Control group.k HepG2 and SK-Hep1 cells were transfected with si RNAs specifically targeting SPOP (si SPOP#1, #2, #3) or negative Control (si NC) for 24 h.WCLs were prepared and SPOP protein levels were determined by Western blot.c Twenty potential E3 ubiquitin ligases targeting BCLAF1 were predicted by the ubibrowser 1.0 database.

a
of the interacting structural domains of BCLAF1 and SPOP, effects of SPOP and BCLAF1 on each other's expression, ubiquitination assay of PD-L1 protein, Western blot of SPOP knockdown, and cellular immunofluorescence to detect BCLAF1 and SPOP expression in HCC cells Diagram of the structure of the SPOP protein.b Schematic representation of SPOP deletion mutants.A binding capacity of SPOP to BCLAF1 is indicated with the symbol.c Western blot of the indicated proteins in WCLs and Co-IP samples of anti-Flag antibody obtained from HEK-293T cells transfected with indicated plasmids.d Western blot of WCLs from HEK-293T cells transfected with indicated plasmids.All quantitation were normalized to the protein levels of GAPDH in Control group.e

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All quantitation were normalized to the protein levels of GAPDH in Control group.l Representative images of Cell immunofluorescence for detection of BCLAF1 and SPOP expression in HepG2 and SK-Hep1 cells.Scale bar, 200 μm.m Quantification of Cell immunofluorescence in (l).All data are shown as mean ± SD (n = 3).***P < 0.001, ns P ≥ 0.05.Supplemental Fig. 3 GST pull-down and ubiquitination of endogenous PD-L1 a Bacterially expressed GST-SPOP or GST bound glutathione-Sepharose beads and incubated with bacterially expressed Flag-BCLAF1 or Flag-BCLAF1-mSBC.Bound Flag-BCLAF1 or Flag-BCLAF1-mSBC was detected by Western blot with anti-Flag antibody.GST and GST-SPOP were detected by Western blot and Coomassie blue staining.b SK-Hep1 cells were treated with MG-132 (20 μM) for 8 h prior to lysis, and in vivo ubiquitination assays were performed using the antibodies shown to detect the effect of BCLAF1-WT and BCLAF1-mSBC on ubiquitination of endogenous PD-Western blot of WCLs from HepG2 and SK-Hep1 cells infected with lentivirus expressing the indicated sh RNAs and BCLAF1-overexpression.All quantitation were normalized to the protein levels of GAPDH in Control group.b Cell colony formation assay of HepG2 and SK-Hep1 cells infected with lentivirus expressing the indicated sh RNA and BCLAF1-overexpression.c, d Statistics of colony number in (b).e, f Cell proliferation assay of HepG2 cells (e) and SK-Hep1 cells (f) infected with lentivirus expressing the indicated sh RNA and BCLAF1-overexpression.g Wound healing assay of HepG2 and SK-Hep1 cells infected with lentivirus expressing the indicated sh RNA and BCLAF1-overexpression.Scale bar, 800 μm.h, i Statistics of the rate of wound healing in (g).j Cell migration assay of HepG2 and SK-Hep1 cells infected with lentivirus expressing the indicated sh RNA and BCLAF1-overexpression.Scale bar, 200 μm.k, l Statistics of the number of cells migrated in (j).m Cell invasion assay of HepG2 and SK-Hep1 cells infected with lentivirus expressing the indicated sh RNA and BCLAF1-overexpression.Scale bar, 200 μm.n, o Statistics of the number of cells invaded in (m).All data are shown as mean ± SD (n = 3).*P < 0.05, **P < 0.01, *Western blot of WCLs from HepG2 and SK-Hep1 cells infected with lentivirus expressing the indicated sh RNAs and transfected with the indicated si RNAs.All quantitation were normalized to the protein levels of GAPDH in Control group.b Cell colony formation assay of HepG2 and SK-Hep1 cells infected with lentivirus expressing the indicated sh RNAs and transfected with the indicated si RNAs.c, d Statistics of colony number in (b).e, f Cell proliferation assay of HepG2 (e) and SK-Hep1 cells (f) infected with lentivirus expressing the indicated sh RNAs and transfected with the indicated si RNAs.g Wound healing assay of HepG2 and SK-Hep1 cells infected with lentivirus expressing the indicated sh RNAs and transfected with the indicated si RNAs.Scale bar, 800 μm.h, i Statistics of the rate of wound healing in (g).j Cell migration assay of HepG2 and SK-Hep1 cells infected with lentivirus expressing the indicated sh RNAs and transfected with the indicated si RNAs.Scale bar, 200 μm.k, l Statistics of the number of cells migrated in (j).m Cell invasion assay of HepG2 and SK-Hep1 cells infected with lentivirus expressing the indicated sh RNAs and transfected with the indicated si RNAs.Scale bar, 200 μm.n, o Statistics of the number of cells invaded in (m).All data are shown as mean ± SD (n = 3).*P < 0.05, **P < 0.01, ***P < 0.001.Supplemental Fig. 6 BCLAF1 induces immune escape of HCC cells partly in an SPOP-PD-L1 axis-dependent manner a HepG2 cells achieving BCLAF1 overexpression, BCLAF1 knockdown, and BCLAF1 knockdown followed by SPOP knockdown were co-cultured with Jurkat cells for 24 h after treatment with DMSO or Atezolizumab (10 ng/mL).Western blot of HepG2 cells in the HCC cell-Jurkat cells co-culture system for detection of PD-L1 expression levels.All quantitation were normalized to the protein levels of GAPDH in Control group.b Flow cytometry analysis of PD-1 binding on the surface of SK-Hep1 cells after treatment of co-culture system with 10 ng/mL Atezolizumab or DMSO for 24 h.c Statistics of mean fluorescence intensity (MFI) for PD-1 in (b).d HepG2 cells achieving BCLAF1 overexpression, BCLAF1 knockdown, and BCLAF1 knockdown followed by SPOP knockdown were co-cultured with Jurkat cells for 24 h after treatment with DMSO or Atezolizumab (10 ng/mL).Apoptosis levels of Jurkat cells were detected by Flow cytometry analysis.e Statistics of apoptotic levels of Jurkat cells in (d).f HepG2 cells achieving BCLAF1 overexpression, BCLAF1 knockdown, and BCLAF1 knockdown followed by SPOP knockdown were co-cultured with Jurkat cells for 24 h after treatment with DMSO or Atezolizumab (10 ng/mL).Cell cycle of Jurkat cells were detected by Flow cytometry analysis.g Statistics of cell cycle of Jurkat cells in (f).h-k HepG2 cells achieving BCLAF1 overexpression, BCLAF1 knockdown, and BCLAF1 knockdown followed by SPOP knockdown were co-cultured with Jurkat cells for 24 h after treatment with DMSO or Atezolizumab (10 ng/mL).The levels of IL-2 (h), IL-4 (i), IL-10 (j), and IFN-γ (k) produced by Jurkat cells were detected by ELISA.All data are shown as mean ± SD (n = 3).*P < 0.05, **P < 0.01, ***P < 0.001.Supplemental Fig. 7 SPOP-binding consensus (SBC) motif-associated BCLAF1 mutant is defective in inducing immune escape of HCC cells a HepG2 cells achieving SPOP knockdown, SPOP overexpression, and exogenous overexpression of BCLAF1-WT and BCLAF1-mSBC after SPOP overexpression were co-cultured with Jurkat cells for 24 h after treatment with DMSO or Atezolizumab (10 ng/mL).Western blot of HepG2 cells in the HCC cell-Jurkat cell co-culture system for detection of PD-L1 expression levels.All quantitation were normalized to the protein levels of GAPDH in Control group.b HepG2 cells achieving SPOP knockdown, SPOP overexpression, and exogenous overexpression of BCLAF1-WT and BCLAF1-mSBC after SPOP overexpression were co-cultured with Jurkat cells for 24 h after treatment with DMSO or Atezolizumab (10 ng/mL).Apoptosis levels of Jurkat cells were detected by Flow cytometry analysis.c Statistics of apoptotic levels of Jurkat cells in (b).d HepG2 cells achieving SPOP knockdown, SPOP overexpression, and exogenous overexpression of BCLAF1-WT and BCLAF1-mSBC after SPOP overexpression were co-cultured with Jurkat cells for 24 h after treatment with DMSO or Atezolizumab (10 ng/mL).Cell cycle of Jurkat cells were detected by Flow cytometry analysis.e Statistics of cell cycle of Jurkat cells in (d).f-i HepG2 cells achieving SPOP knockdown, SPOP overexpression, and exogenous overexpression of BCLAF1-WT and BCLAF1-mSBC after SPOP overexpression were co-cultured with Jurkat cells for 24 h after treatment with DMSO or Atezolizumab (10 ng/mL).The levels of IL-2 (f), IL-4 (g), IL-10 (h), and IFN-γ (i) produced by Jurkat cells were detected by ELISA.All data are shown as mean ± SD (n = 3).**P < 0.01, ***P < 0.001.Supplemental Fig. 8 Effect of SPOP on the expression levels of HIF-1α and BCLAF1, the effect of BCLAF1 on SPOP-PD-L1 interaction, and SPOP mediates the indirect interaction between BCLAF1 and PD-L1 Western blot of WCLs from SK-Hep1 cells treated with 200 µM Cocl2 24 h.All quantitation were normalized to the protein levels of GAPDH in the untransfected Myc-SPOP plasmid group.b, c qRT-PCR measurement of BCLAF1 (b) and HIF-1α (c) mRNA levels in SK-Hep1 cells treated with 200 µM Cocl2 24 h.d, e Western blot of the indicated proteins in WCLs and Co-IP samples of anti-Flag antibody obtained from HEK-293T cells transfected with indicated plasmids.All data are shown as mean ± SD (n = 3).ns P ≥ 0.05.Supplemental Fig. 9 Predicted upstream regulators of BCLAF1 at transcriptional, post-transcriptional, and post-translational levels a Ten potential transcription factors targeting BCLAF1 predicted by GRTD, HumanTFDB, and PROMO databases.b Five potential microRNAs (miRNAs) targeting BCLAF1 predicted by miRDB, mirDIP, miRWalk, and TargetScan databases.

Table 1 .
424 clinical characteristics of HCC patients from TCGA.

Table 7 .
Primers and si RNA/sh RNA sequence information.