Stabilization of Pin1 by USP34 promotes Ubc9 isomerization and protein sumoylation in glioma stem cells

The peptidyl-prolyl cis-trans isomerase Pin1 is a pivotal therapeutic target in cancers, but the regulation of Pin1 protein stability is largely unknown. High Pin1 expression is associated with SUMO1-modified protein hypersumoylation in glioma stem cells (GSCs), but the underlying mechanisms remain elusive. Here we demonstrate that Pin1 is deubiquitinated and stabilized by USP34, which promotes isomerization of the sole SUMO E2 enzyme Ubc9, leading to SUMO1-modified hypersumoylation to support GSC maintenance. Pin1 interacts with USP34, a deubiquitinase with preferential expression and oncogenic function in GSCs. Such interaction is facilitated by Plk1-mediated phosphorylation of Pin1. Disruption of USP34 or inhibition of Plk1 promotes poly-ubiquitination and degradation of Pin1. Furthermore, Pin1 isomerizes Ubc9 to upregulate Ubc9 thioester formation with SUMO1, which requires CDK1-mediated phosphorylation of Ubc9. Combined inhibition of Pin1 and CDK1 with sulfopin and RO3306 most effectively suppresses orthotopic tumor growth. Our findings provide multiple molecular targets to induce Pin1 degradation and suppress hypersumoylation for cancer treatment.

b Coomassie blue staining (left) and mass spectrometry identification (right) of immunoprecipitation products containing the Pin1-interacting proteins.T4121 GSC lysate was subjected to immunoprecipitation with IgG or anti-Pin1 antibodies followed by gel electrophoresis and Coomassie blue staining.The heavy chain, light chain, and the band of Pin1 bait were cut off the gel, and the rest proteins were subjected to mass spectrometry analysis.The top 10 identified proteins were listed on the right.c Representative images of immunofluorescent analysis of USP34 (red) and Pin1 (green) in human primary GBMs.Frozen sections of human GBMs were immunostained with antibodies against USP34 and Pin1, and counterstained with Hoechst to show nuclei (blue).Scale bar, 20 μm.The experiment was repeated for 5 times with similar results.
e Immunoblot analysis of USP34 and Pin1 protein levels in T387 GSCs expressing shUSP34 or shNT.
f Immunoblot analysis of the polyubiquitination of endogenous Pin1 proteins in T387 GSCs expressing shUSP34.Cells were harvested 72 hours post-lentiviral infection.MG132 (10 μM) was added to cell culture 6 hours before harvest for the ubiquitination assay.Cell lysate was subjected to immunoprecipitation with anti-Pin1 antibodies followed by immunoblot with anti-Ub antibodies.
g Immunoblot analysis of the protein levels of the wild type Pin1 (WT) and the Pin1-K117A mutant in T387 GSCs expressing shUSP34.Ectopic flag-tagged Pin1 proteins were expressed in GSCs through lentiviral infection.Cells were further infected with shUSP34 lentiviruses and harvested 72 hours post infection.
The blotting experiments were repeated at least three times with biological replicates (a, e-g).
Source data are provided as a Source Data file.e Representative images of tumorsphere formation of T387 GSCs expressing shUSP34 or shNT.Twenty-four hours after lentiviral infection, 2,000 cells were planted in each well of 96well plates and cultured for 5 days.Scale bar, 50 μm.f, g Statistical quantifications of tumorsphere numbers (f) and sizes (g) of T387 GSCs expressing shUSP34 or shNT.Three random 20× fields were used for calculation.(n = 3 biological replicates; ***, P < 0.001; mean ± s.d.; two tailed unpaired t-test).
The blotting experiments were repeated at least three times with biological replicates (d).
Source data are provided as a Source Data file.The blotting experiments were repeated at least three times with biological replicates (a, d-j).
Source data are provided as a Source Data file.Source Data -Supplementary Figure 7

a
Immunoblot analysis of Pin1 protein levels in GSCs and NSTCs treated with MG132 (10 μM) or DMSO for 6 hours.