Immune checkpoint blockade (ICB), particularly anti-PD-1/PD-L1 therapy, has emerged as an important treatment option for bladder cancer (BLCA). However, a substantial proportion of patients fail to achieve durable clinical benefit due to primary or acquired resistance. Accumulating evidence indicates that an immunosuppressive, non-inflamed tumor microenvironment (TME) is a major determinant of resistance to immunotherapy. Nevertheless, the key tumor-intrinsic regulators that shape the immune landscape and limit ICB efficacy in BLCA remain poorly defined. Integrated multi-omics analyses were performed using bulk RNA sequencing, single-cell RNA sequencing, and publicly available and in-house BLCA cohorts to characterize the immunological role of RNF128. Bioinformatic analyses were applied to assess immune cell infiltration, immune signaling pathways, and molecular subtypes. Functional validation was conducted using RNF128 knockdown and overexpression in human and murine bladder cancer cell lines, chemotaxis assays, T cell–mediated cytotoxicity assays, and syngeneic mouse tumor models combined with anti-PD-1 therapy. The association between RNF128 expression and ICB response was further evaluated in independent immunotherapy cohorts. RNF128 was highly expressed in bladder cancer and was associated with an immunosuppressive TME. Elevated RNF128 expression was associated with reduced CD8⁺ T-cell infiltration, lower expression of T-cell-recruiting chemokines, including CCL3, CCL4, CCL5, CXCL9, and CXCL10, and lower T cell–inflamed signature scores. Single-cell analyses revealed that RNF128 was predominantly expressed in malignant urothelial cells and was associated with lower enrichment of cytokine- and chemokine-related transcriptional signatures. Functional experiments showed that manipulation of RNF128 affected CD8⁺ T-cell recruitment and cytotoxic activity, whereas RNF128 knockdown enhanced the antitumor efficacy of PD-1 blockade in a syngeneic mouse model. Clinically, lower RNF128 expression was associated with more favorable ICB responses and improved survival outcomes in the evaluated immunotherapy cohorts. RNF128 is associated with a non-inflamed immune microenvironment and reduced ICB responsiveness in BLCA, while functional studies support a role for tumor-cell RNF128 in limiting CD8⁺ T-cell recruitment and antitumor activity. RNF128 may therefore represent a candidate biomarker and potential therapeutic target for improving immunotherapy response in BLCA.
Mitochondrial fission and altered Complex II expression and function are hallmarks of Huntington’s (HD) and Alzheimer’s (AD) diseases. Notably, peroxisomal fission protein, PEX11β, works in coordination with mitochondrial fission proteins and exhibits a unique co-expression pattern with respiratory chain complexes. However, whether a PEX11β-mediated shared mechanism drives mitochondrial fragmentation across different neurodegenerative conditions remains unclear. Using patient transcriptomic data and in vitro cellular models, we establish a novel connection between disease-associated downregulation of PEX11β, mitochondrial fragmentation, and impaired expression of fusion and Complex II genes. Restoration of PEX11β alleviates motor and cognitive deficits, and reinstates mitochondrial gene expression in the striatum of 3-NP-induced HD mice. In silico analysis and ChIP-PCR reveal Yin Yang 1 is a critical transcription factor regulating co-expression of PEX11β and Complex II genes. Our findings uncover PEX11β-mediated regulation of mitochondrial fission-fusion dynamics, linking mitochondrial integrity to behavioral outcomes, and offer a novel insight into amyloid-associated neuropathology.
Alternative splicing of the CSNK1A1 gene generates multiple CK1α isoforms with distinct subcellular localizations and regulatory properties. While cytoplasmic CK1α is a well-established negative regulator of canonical WNT signaling, the role of nuclear CK1α remains poorly understood. Here, we identify L-insert-containing nuclear CK1α isoforms as functional components of the β-catenin/TCF transcriptional complex in gastrointestinal cancer cells. Using isoform-specific assays, we demonstrate that CK1α isoforms containing the L-insert exhibit greater nuclear accumulation than isoforms lacking the L-insert and support WNT-driven transcription. Knockdown of L-insert-containing CK1α isoforms in esophageal adenocarcinoma (EAC) and colorectal cancer (CRC) models reduces β-catenin recruitment to TCF-bound chromatin and attenuates WNT target gene expression. Kinase-inactive CK1α mutants retained their association with the β-catenin/TCF complex but showed reduced WNT reporter activity, indicating that CK1α catalytic activity is dispensable for complex association but is required for full transcriptional activation. Consistent with the canonical negative regulatory function of CK1α, pharmacological activation of CK1α with SSTC3 increased β-catenin Ser45 phosphorylation, reduced active β-catenin abundance, and limited its nuclear availability and binding to TCF sites, thereby attenuating WNT transcriptional activity and reducing tumor-initiating cell (TIC)-enriched populations in gastrointestinal tumors. These effects translate into reduced tumorigenic potential and delayed tumor regrowth in vivo. Together, our findings identify L-insert-containing nuclear CK1α isoforms as positive regulators of WNT transcription, in contrast to the negative regulatory function of cytoplasmic CK1α within the β-catenin destruction complex, and provide a rationale for pharmacological CK1α activation as a strategy to target WNT-driven TICs.
P-glycoprotein (P-gp/MDR1) overexpression in macrophages contributes to tuberculosis (TB) treatment failure by reducing intracellular drug accumulation. The molecular mechanisms by which host macrophages upregulate P-gp remain poorly defined. The present study investigated whether CK2α functions as an upstream kinase regulating P-gp expression in macrophages and examined its therapeutic relevance in TB. P-gp regulation was characterized in PMA-differentiated THP-1 macrophages by immunoblotting, RT-PCR, qRT-PCR, immunofluorescence, co-immunoprecipitation, and subcellular fractionation, and in U-937 macrophages by immunoblotting. Signaling pathways were dissected using pharmacological inhibitors, siRNA knockdown, and overexpression. P-gp efflux function was assessed by rhodamine-123 accumulation assay. Flow cytometry was used to evaluate CK2α-dependent P-gp regulation in PBMC-derived primary macrophages. P-gp expression and TB pathology were examined in Mycobacterium tuberculosis H37Rv-infected BALB/c mice. During monocyte-to-macrophage differentiation, CK2α activated AKT at Ser129, leading to NF-κB-dependent P-gp induction. In mature macrophages, rifampin induced P-gp through CK2α-mediated Hsp90β phosphorylation and subsequent PXR stabilization. CK2 inhibition or CSNK2A1 knockdown suppressed P-gp expression through both pathways and increased intracellular rhodamine-123 accumulation. In Mtb-infected mice, co-administration of the CK2 inhibitor DRB with rifampin suppressed lung P-gp expression, reduced pulmonary bacterial burden compared to rifampin monotherapy, and attenuated lung inflammation. PBMC-derived primary macrophages showed the same CK2α-dependent dual pathway regulation. The present study identifies CK2α as a common upstream kinase for both NF-κB- and PXR-dependent P-gp induction in macrophages. CK2 inhibitors, for which clinical safety data are available from oncology trials, may warrant evaluation as adjunctive agents against P-gp-mediated drug resistance in TB treatment.
Endosomal dysfunction is increasingly recognised as an early event in Alzheimer’s disease. Hyperactivation of the early endosomal regulator Rab5 has been implicated in early AD-associated pathology, but its downstream signalling consequences and therapeutic relevance remain incompletely defined. We investigated whether Rab5-associated endo-lysosomal abnormalities are linked to oxytocin receptor trafficking defects and impaired receptor-associated signalling in early Alzheimer’s disease-relevant settings. Young APP/PS1 mice were used to assess endogenous endo-lysosomal abnormalities in the hippocampus. A hippocampus-targeted Rab5 overactivation model was established by stereotaxic delivery of AAV-hSyn-EGFP-Rab5Q79L to test whether sustained Rab5 activation was sufficient to induce similar alterations. Oxytocin receptor localisation, endosomal marker colocalisation, and OTR-associated downstream signalling readouts were evaluated by confocal immunofluorescence and western blotting. Receptor internalisation/recycling assays, membrane protein western blotting, OT rechallenge, and OTR antagonist experiments were performed in HEK293T cells to assess plasma-membrane OTR recovery and OT-responsive ERK/CREB phosphorylation. RNA sequencing, synaptic marker analysis, resting-state functional magnetic resonance imaging, structural magnetic resonance imaging, and behavioural testing were used to assess downstream molecular, circuit-level, and functional consequences. Group comparisons were analysed using one-way analysis of variance followed by Tukey’s post hoc test. Young APP/PS1 mice exhibited enlarged Rab5-positive early endosomal structures, increased overlap of Rab5 with Rab7 and LAMP1, abnormal intracellular redistribution of the oxytocin receptor, and reduced phosphorylation of ERK and CREB in the hippocampus. Rab5 overactivation reproduced Rab5-associated endosomal abnormalities and was accompanied by altered OTR endosomal association, impaired plasma-membrane OTR recovery, and reduced OT-evoked ERK/CREB phosphorylation. These receptor-level and signalling changes were accompanied by disruption of neuronal signalling-related transcriptional programmes, loss of excitatory and inhibitory synapses, reduced spontaneous brain activity, weakened hippocampal-medial prefrontal cortex functional connectivity, hippocampal atrophy, and cognitive impairment. Across the two models, MW150 treatment was associated with partial improvement in endosomal abnormalities, OTR-associated signalling readouts, and behavioural measures; additional synaptic and imaging improvements were observed in the Rab5-overactivation model. Rab5-associated endo-lysosomal trafficking abnormalities are accompanied by altered oxytocin receptor localisation, reduced OTR-associated signalling readouts, and multi-level early AD-relevant phenotypes. The partial improvement in molecular, synaptic, network, and behavioral abnormalities by MW150 supports the modifiability of the p38α–Rab5/endosomal axis. At the same time, further work is needed to establish whether OTR trafficking directly mediates these downstream phenotypes.
