MPP8 inhibits PRKN signaling through interactions with SIRT1 and ZEB1 to promote bladder cancer proliferation and metastasis

Background: Metastatic bladder cancer (mBC) remains incurable, and epigenetic dysregulation is associated with disease progression. We hypothesize that the H3K9me3 reader protein MPP8 complexes with class III histone deacetylase SIRT1 and transcription factor ZEB1 to drive proliferation and metastasis. Here, we explore how MPP8 knockdown (MPP8 KD ) disrupts MPP8-SIRT1-ZEB1 interactions, thereby altering differentially expressed genes (DEGs) and gene networks and abrogating proliferation and metastasis. Methods: UM-UC-3 mBC cells were used to compare MPP8 KD and untransfected (MPP8 WT ) cells. RNA-seq quantified differential expression. DEGs were analyzed in R v4.1.1 using DESeq2.3. Gene enrichment was assessed with GSEA v4.2.3 (Hallmark sets) and IPA pathway analyses (Qiagen). Co-IP WBs evaluated MPP8-SIRT1-ZEB1 protein complexes. CellTiter-Glo™ evaluated differences in viability. Clonogenic assays assessed proliferation while migration/invasion assays evaluated metastatic potential. MPP8 KD and MPP8 WT cells were injected into NSG mouse flanks, and changes to both tumor volume and TBW were measured until tumors were ≥ 2 cm 3 . Results: Over-representation analysis of DEGs indicated MPP8 KD reduced mBC cell growth and DNA replication, as well as increased inhibition of cell migration. GSEA identified downregulated E2F (NES=-1.9, q=0.02) and enrichment of TNFalpha signaling via NFKB gene sets (NES=1.71, q=0.01). IPA revealed activation of the Parkinson's signaling pathway (z-score=1.54, P=1.1E-03), and consistent with GSEA analyses, predicted reduced proliferation and cellular stress responses. A custom IPA gene network of top DEGs included Parkinson's signaling pathway genes (e.g., PRKN) and demonstrated that MPP8, SIRT1, ZEB1 all act upstream of pathway genes. Moreover, SIRT1 and PRKN were predicted to interact through protein-protein interactions, while MPP8 KD directly increased PRKN expression (LFC=1.658, q=2.39E-03). Functionally, we demonstrated MPP8 forms a complex with SIRT1 and ZEB1 in MPP8 WT cells, while the MPP8-SIRT1-ZEB1 interactions were lost in MPP8 KD cells. MPP8 KD reduced viability (44% vs. 84% after 48 h; P=0.0001), colony growth (13% vs. 36% plate covered with colonies at 10 d; P=0.0001), number of migrated cells (97 vs. 470 cells after 18 h; P<0.0001), and number of invading cells (44 vs. 130 cells after 18 h; P=0.02). By D+32 post-injection, all mice with MPP8 WT cells were sacrificed (mean vol. 2,859 ± 761 mm 3 ; n=8). At D+32, tumor volume was significantly smaller in MPP8 KD1 (mean vol. 234 ± 447 mm 3 , P<0.0001; n=8) and MPP8 KD2 mice (mean vol. 1,341 ± 785 mm 3 , P=0.002; n=8). At D+32, changes in TBW were not observed. Conclusions: Our data suggest the MPP8-SIRT1-ZEB1 axis is a key driver of mBC, and MPP8 KD results in PRKN activation that could halt mBC proliferation and metastasis.