CD73, an ecto-5′-nucleotidase (NT5E), serves as an immune checkpoint by generating adenosine (ADO), which suppresses immune activation through the A_2A receptor. Elevated CD73 levels in tumor tissues correlate with poor clinical outcomes. However, the crucial source of CD73 activity within the tumor microenvironment remains unspecified. Here, we demonstrate that cancer-associated fibroblasts (CAFs) constitute the prominent CD73^hi population in human colorectal cancers (CRCs) and two CD73⁻ murine tumor models, including a modified CRC. Clinically, high CAF abundancy in CRC tissues correlates strongly with elevated CD73 activity and poor prognosis. Mechanistically, CAF-CD73 expression is enhanced via an ADO-A_2B receptor-mediated feedforward circuit triggered by tumor cell death, which enforces the CD73-checkpoint. Simultaneous inhibition of A_2A and A_2B pathways with CD73-neutralization synergistically enhances antitumor immunity in CAF-rich tumors. Therefore, the strategic and effective targeting of both the A_2B-mediated ADO-CAF-CD73 feedforward circuit and A_2A-mediated immune suppression is crucial for improving therapeutic outcomes.

Although clinically apparent metastasis is associated with late stages of cancer development, micro-metastatic dissemination may be an early event. However, the fate of these early disseminated tumor cells (DTC) remains elusive. We show that despite their capacity to disseminate into secondary organs, 4T1 tumor models develop overt metastasis while EMT6-tumor bearing mice clear DTCs shed from primary tumors as well as those introduced by intravenous (IV) injection. Following the surgical resection of primary EMT6 tumors, mice do not develop detectable metastasis and reject IV-injected tumor cells. In contrast, these cells readily grow and metastasize in immuno-deficient athymic or Rag2^−/− mice, an effect mimicked by CD8⁺ T-cell depletion in immunocompetent mice. Furthermore, recombinant G-CSF or adoptive transfer of granulocytic-MDSCs isolated from 4T1 tumor-bearing mice, induce metastasis by suppressing CD8⁺ T-cells in EMT6-primed mice. Our studies support the concept of immune surveillance providing molecular insights into the immune mechanisms during tumor progression.

Purpose: The perturbation of metabolic pathways in high-grade bladder cancer has not been investigated. We aimed to identify a metabolic signature in high-grade bladder cancer by integrating unbiased metabolomics, lipidomics, and transcriptomics to predict patient survival and to discover novel therapeutic targets. Experimental Design: We performed high-resolution liquid chromatography mass spectrometry (LC-MS) and bioinformatic analysis to determine the global metabolome and lipidome in high-grade bladder cancer. We further investigated the effects of impaired metabolic pathways using in vitro and in vivo models. Results: We identified 519 differential metabolites and 19 lipids that were differentially expressed between low-grade and high-grade bladder cancer using the NIST MS metabolomics compendium and lipidblast MS/MS libraries, respectively. Pathway analysis revealed a unique set of biochemical pathways that are highly deregulated in high-grade bladder cancer. Integromics analysis identified a molecular gene signature associated with poor patient survival in bladder cancer. Low expression of CPT1B in high-grade tumors was associated with low FAO and low acyl carnitine levels in high-grade bladder cancer, which were confirmed using tissue microarrays. Ectopic expression of the CPT1B in high-grade bladder cancer cells led to reduced EMT in in vitro, and reduced cell proliferation, EMT, and metastasis in vivo. Conclusions: Our study demonstrates a novel approach for the integration of metabolomics, lipidomics, and transcriptomics data, and identifies a common gene signature associated with poor survival in patients with bladder cancer. Our data also suggest that impairment of FAO due to downregulation of CPT1B plays an important role in the progression toward high-grade bladder cancer and provide potential targets for therapeutic intervention.

O-GalNAc glycans (or mucin O-glycans) play pivotal roles in diverse biological and pathological processes, including tumor growth and progression. Structurally defined O-GalNAc glycans are essential for functional studies but synthetic challenges and their inherent structural diversity and complexity have limited access to these compounds. Herein, we report an efficient and robust chemoenzymatic modular assembly (CEMA) strategy to construct structurally diverse O-GalNAc glycans. The key to this strategy is the convergent assembly of O-GalNAc cores 1–4 and 6 from three chemical building blocks, followed by enzymatic diversification of the cores by 13 well-tailored enzyme modules. A total of 83 O-GalNAc glycans presenting various natural glycan epitopes are obtained and used to generate a unique synthetic mucin O-glycan microarray. Binding specificities of glycan-binding proteins (GBPs) including plant lectins and selected anti-glycan antibodies towards these O-GalNAc glycans are revealed by this microarray, promoting their applicability in functional O-glycomics. Serum samples from colorectal cancer patients and healthy controls are assayed using the array reveal higher bindings towards less common cores 3, 4, and 6 than abundant cores 1 and 2, providing insights into O-GalNAc glycan structure-activity relationships.

Background: The current system to predict the outcome of smokers with bladder cancer is insufficient due to complex genomic and transcriptomic heterogeneities. This study aims to identify serum metabolite-associated genes related to survival in this population. Methods: We performed LC/MS-based targeted metabolomic analysis for >300 metabolites in serum obtained from two independent cohorts of bladder cancer never smokers, smokers, healthy smokers, and healthy never smokers. A subset of differential metabolites was validated using Biocrates absoluteIDQ p180 Kit. Genes associated with differential metabolites were integrated with a publicly available cohort of The Cancer Genome Atlas (TCGA) to obtain an intersecting signature specific for bladder cancer smokers. Results: Forty metabolites (FDR < 0.25) were identified to be differential between bladder cancer never smokers and smokers. Increased abundance of amino acids (tyrosine, phenylalanine, proline, serine, valine, isoleucine, glycine, and asparagine) and taurine were observed in bladder cancer smokers. Integration of differential metabolomic gene signature and transcriptomics data from TCGA cohort revealed an intersection of 17 genes that showed significant correlation with patient survival in bladder cancer smokers. Importantly, catechol-O-methyltransferase, iodotyrosine deiodinase, and tubulin tyrosine ligase showed a significant association with patient survival in publicly available bladder cancer smoker datasets and did not have any clinical association in never smokers. Conclusions: Serum metabolic profiling of bladder cancer smokers revealed dysregulated amino acid metabolism. It provides a distinct gene signature that shows a prognostic value in predicting bladder cancer smoker survival. Impact: Serum metabolic signature–derived genes act as a predictive tool for studying the bladder cancer progression in smokers.

Cumulative evidence suggests that the heat shock protein 90 (HSP90) co-chaperone UNC-45 myosin chaperone A (UNC45A) contributes to tumorigenesis and that its expression in cancer cells correlates with proliferation and metastasis of solid tumors. However, the molecular mechanism by which UNC45A regulates cancer cell proliferation remains largely unknown. Here, using siRNA-mediated gene silencing and various human cells, we report that UNC45A is essential for breast cancer cell growth, but is dispensable for normal cell proliferation. Immunofluorescence microscopy, along with gene microarray and RT-qPCR analyses, revealed that UNC45A localizes to the of cancer cell nucleus, where it up-regulates the transcriptional activity of the glucocorticoid receptor and thereby promotes expression of the mitotic kinase NIMA-related kinase 7 (NEK7). We observed that UNC45A-deficient cancer cells exhibit extensive pericentrosomal material disorganization, as well as defects in centrosomal separation and mitotic chromosome alignment. Consequently, these cells stalled in metaphase and cytokinesis and ultimately underwent mitotic catastrophe, phenotypes that were rescued by heterologous NEK7 expression. Our results identify a key role for the co-chaperone UNC45A in cell proliferation and provide insight into the regulatory mechanism. We propose that UNC45A represents a promising new therapeutic target to inhibit cancer cell growth in solid tumor types.