Journal: Molecular Cancer
Article Title: CDK6/4 inactivates BAP1 deubiquitinase destabilizing VHL to promote metastatic colonization in liver
doi: 10.1186/s12943-026-02650-5
Figure Lengend Snippet: BAP1 impedes hepatic colonization of UM cells via VHL. A -C Inhibition of CDK6/4 activity escalates the expression of endogenous VHL protein. Mel270 and Omm2.3 cells were incubated with increasing concentrations of Palbociclib for 24 h. The expression of BAP1 and VHL was analyzed by Western blotting ( A ) and qRT-PCR ( B ), respectively. Mel270 cells were incubated with 100 mg/ml of CHX for durations as indicated in the presence of Palbociclib (10.0 µmol/L, 16 h) or not. Cell lysates were then subjected to Wester blotting analysis with anti-VHL ( C ). D Inhibition of CDK 4/6 decreases the ubiquitination of VHL in Mel270 cells. Mel270 cells transfected with constructs of His-Ub and HA-VHL were exposed to Palbociclib (10.0 µmol/L) for 18 h in presence of MG132. His-tagged ubiquitin was immunoprecipitated with Ni-NTA beads and subjected to immunoblotting analysis with anti-HA. E-F Palbociclib inhibits the growth of UM cells. UM cells and the normal retinal pigment epithelia (RPE) cell line ARPE-19 were treated with increasing concentrations of Palbociclib for 72 h. Cell viability was analyzed by MTS assay. IC 50 values are 15.88 µmol/L for 92.1, 11.67 µmol/L for Mel270, 19.97 µmol/L for Omm2.3, 15.48 µmol/L for Omm1, and 100 µmol/L for ARPE-19, respectively ( E ). After treated with Palbociclib for 24 h, UM cells were harvested and inoculated in double layer soft agar culture for colony formation assay. IC 50 values are 2.59 µmol/L for 92.1, 2.51 µmol/L for Mel270, 3.35 µmol/L for Omm2.3, and 3.71 µmol/L for Omm1, respectively ( F ). G-H Palbociclib eliminates the self-renewal of UM stem-like cells. After incubated with Palbociclib (5.0 µmol/L, 24 h), Mel270 and Omm2.3 cells were subjected to flow cytometer analysis for ALDH + percentage ( G ) and serial culture of tumorsphere ( H ). Data are mean ± SD, **, p < 0.01; ***, p < 0.001; ****, p <0.0001, Student’s t test. I-L Palbociclib suppresses the hepatic colonization of UM cells. Mel270-luc and Omm2.3-luc cells were intrasplenically injected in NOG mice. The mice were orally administered with control or Palbociclib (100 mg/kg/d) for 4 ~ 5 weeks. The spreading of UM cells was monitored by BLI signals. The representative photos of BLI signals in mice on day 35 are shown ( I ). The curves of BLI signals were plotted against days after treatment. Data are mean ± SD. **, p < 0.01; ***, p < 0.001, comparisons were made by the Student’s t test between treatment on the last day ( J ). The photos of livers bearing Mel270-luc or Omm2.3-luc metastatic nodules are shown. The nodules on the liver surface were compared between placebo and Palbociclib treatment. Data are mean ± SD. ***, p < 0.001; ****, p < 0.0001, Student’s t test ( K ). The metastatic foci of UM cells in liver sections were analyzed by H&E staining ( L ). Scale bar, 500 μm ( top ); 200 μm ( bottom )
Article Snippet: Antibody against BAP1 (cat#A302-243A, RRID: AB_1731059) was from Bethyl (Montgomery, Texas).
Techniques: Inhibition, Activity Assay, Expressing, Incubation, Western Blot, Quantitative RT-PCR, Ubiquitin Proteomics, Transfection, Construct, Immunoprecipitation, MTS Assay, Colony Assay, Flow Cytometry, Injection, Control, Staining
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![BAP1 is phosphorylated at Ser369 by CDK6/4. A Immunoprecipitation of cell lysates from Mel270 or Omm2.3 with IgG or anti-BAP1 antibody was immunoblotted with antibodies against CDK4 and CDK6, respectively. B His-CDK4 or His-CDK6 were expressed by pET-28a plasmid in bacteria. The recombinant GST and GST-BAP1 proteins purified from bacteria expressing pGEX-4T1 constructs were in vitro incubated with the recombinant His-CKD6 ( left ) or His-CDK4 ( right ) protein purified from bacteria expressing pET-28a constructs. The protein interaction between BAP1 and CDK6/4 was analyzed by GST pull-down assay. C-D 293T cells were transfected with pIRES and pIRES-BAP1-Flag. Cell lysates were immunoprecipitated with S-protein agarose beads and subjected to immunoblotting analysis with antibodies against CDK4, CDK6 ( C ) and the antibody specific to pan-phospho-CDK substrates ( D ), respectively. E 293T cells were transfected with pIRES and pIRES-Flag-WT BAP1, and pIRES-Flag-BAP1 SA mutants. Cell lysates were immunoprecipitated with S-protein agarose beads and subjected to immunoblotting with the antibody specific to phospho-CDK substrates. F Mel270 cells transfected with plasmids encoding pLV3, pLV3-Flag-BAP1 WT and pLV3-Flag-BAP1 S369A mutant were treated with LY2835219 (10 µmol/L) for 24 h. Cell lysates were immunoprecipitated with S-protein agarose beads and subjected to immunoblotting with the antibody specific to the phosphorylated BAP1 at S369. G Phosphorylation of BAP1 at Ser369 retards polyubiquitylation of VHL. 293T cells stably transduced with scramble or BAP1-shRNA were transfected with pLV3-HA-VHL and pIRES-BAP1 [WT or its mutants (S369A, S369D)], followed by incubation with MG132 for 6 h. Cell lysates were immunoprecipitated with HA beads and subjected to Western blotting analysis with anti-ubiquitin for the polyubiquitylated VHL protein. H Phosphorylation of BAP1 at Ser369 elevated the expression of VHL. After transduced with scramble or BAP1 shRNA, Mel270-luc and Omm2.3-luc cells were restored with pIRES-BAP1 WT or its mutants (S369A, S369D). Cell lysates were subjected to Western blotting analysis with anti-BAP1 and anti-VHL, respectively. I-J After transduced with lentiviral scramble or shCDK6/4, whole cell lysates ( I ) and exosomes ( J ) from HCT116 cells were then subjected to Western blotting with the specific antibodies against CDK6 and CDK4, respectively. K After exposed to exosomes purified from the CDK6/4-depleted HCT116 cells for 24 h, HCT116 cells were subjected to Western blotting analysis. L-N HCT116 cells were cultured in the presence of TGF-β1 for 24 h, the EMT cells were analyzed ( L ). Data are mean ± SD, *, p < 0.05; ***, p < 0.001, one-way ANOVA, post hoc intergroup comparisons. Whole cell lysates ( M ) and exosomes ( N ) from HCT116 cells were subjected to Western blotting analysis. O MC38 cells were cultured in complete medium, or pH 6.5 complete medium, or 5% O 2 for 48 h, lysates of exosomes fractionated in the culture medium were subjected to Western blotting](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_2817/pmc13202817/pmc13202817__12943_2026_2650_Fig5_HTML.jpg)
