Inferring Bladder Cancer Evolution from Mucosal Field Effects by Whole-Organ Spatial Mutational, Proteomic, and Metabolomic Mapping.

Multi-platform mutational, proteomic, and metabolomic spatial mapping was used on the whole-organ scale to identify the molecular evolution of bladder cancer from mucosal field effects. We identified complex proteomic and metabolomic dysregulations in microscopically normal areas of bladder mucosa adjacent to dysplasia and carcinoma in situ . The mutational landscape developed in a background of complex defects of protein homeostasis which included dysregulated nucleocytoplasmic transport, splicesome, ribosome biogenesis, and peroxisome. These changes were combined with altered urothelial differentiation which involved lipid metabolism and protein degradations controlled by PPAR. The complex alterations of proteome were accompanied by dysregulation of gluco-lipid energy-related metabolism. The analysis of mutational landscape identified three types of mutations based on their geographic distribution and variant allele frequencies. The most common were low frequency α mutations restricted to individual mucosal samples. The two other groups of mutations were associated with clonal expansion. The first of this group referred to as ß mutations occurred at low frequencies across the mucosa. The second of this group called γ mutations increased in frequency with disease progression. Modeling of the mutations revealed that carcinogenesis may span nearly 30 years and can be divided into dormant and progressive phases. The α mutations developed gradually in the dormant phase. The progressive phase lasted approximately five years and was signified by the advent of ß mutations, but it was driven by γ mutations which developed during the last 2-3 years of disease progression to invasive cancer. Our study indicates that the understanding of complex alterations involving mucosal microenvironment initiating bladder carcinogenesis can be inferred from the multi-platform whole-organ mapping.

The Hepatokine Orosomucoid 2 Mediates Beneficial Metabolic Effects of Bile Acids.

Bile acids (BAs) are pleiotropic regulators of metabolism. Elevated levels of hepatic and circulating BAs improve energy metabolism in peripheral organs, but the precise mechanisms underlying the metabolic benefits and harm still need to be fully understood. In the current study, we identified orosomucoid 2 (ORM2) as a liver-secreted hormone (i.e., hepatokine) induced by BAs and investigated its role in BA-induced metabolic improvements in mouse models of diet-induced obesity. Contrary to our expectation, under a high-fat diet (HFD), our Orm2 knockout (Orm2-KO) exhibited a lean phenotype compared with C57BL/6J control, partly due to the increased energy expenditure. However, when challenged with a HFD supplemented with cholic acid, Orm2-KO eliminated the antiobesity effect of BAs, indicating that ORM2 governs BA-induced metabolic improvements. Moreover, hepatic ORM2 overexpression partially replicated BA effects by enhancing insulin sensitivity. Mechanistically, ORM2 suppressed interferon-γ/STAT1 activities in inguinal white adipose tissue depots, forming the basis for anti-inflammatory effects of BAs and improving glucose homeostasis. In conclusion, our study provides new insights into the molecular mechanisms of BA-induced liver-adipose cross talk through ORM2 induction.

Serum Extracellular Vesicle Protein Profiling for Prediction of Corneal Transplant Rejection.

BACKGROUND: Corneal transplantation is the most common transplant procedure worldwide. Despite immune and angiogenic privilege of the cornea, 50% to 70% of corneal transplants fail in high-risk recipients, primarily because of immune rejection. Therefore, it is crucial to identify predictive biomarkers of rejection to improve transplant survival. METHODS: In search for predictive biomarkers, we performed proteomics analysis of serum extracellular vesicles (EVs) in a fully major histocompatibility complex-mismatched (C57BL/6-to-BALB/c) murine corneal transplantation model, wherein 50% of transplants undergo rejection by day 28 following transplantation. RESULTS: Our time course study revealed a decrease in the number of serum EVs on day 1, followed by a gradual increase by day 7. A comparative analysis of proteomics profiles of EVs from transplant recipients with rejection (rejectors) and without rejection (nonrejectors) found a distinct enrichment of histocompatibility 2, Q region locus 2, which is a part of major histocompatibility complex-class I of donor C57BL/6 mice, in day 7 EVs of rejectors, compared with nonrejectors, syngeneic controls, or naïve mice. In contrast, serum amyloid A2, a protein induced in response to injury, was increased in day 7 EVs of nonrejectors. CONCLUSIONS: Our findings offer noninvasive EV-based potential biomarkers for predicting corneal allograft rejection or tolerance.

Organelle proteomic profiling reveals lysosomal heterogeneity in association with longevity.

Lysosomes are active sites to integrate cellular metabolism and signal transduction. A collection of proteins associated with the lysosome mediate these metabolic and signaling functions. Both lysosomal metabolism and lysosomal signaling have been linked to longevity regulation; however, how lysosomes adjust their protein composition to accommodate this regulation remains unclear. Using deep proteomic profiling, we systemically profiled lysosome-associated proteins linked with four different longevity mechanisms. We discovered the lysosomal recruitment of AMP-activated protein kinase and nucleoporin proteins and their requirements for longevity in response to increased lysosomal lipolysis. Through comparative proteomic analyses of lysosomes from different tissues and labeled with different markers, we further elucidated lysosomal heterogeneity across tissues as well as the increased enrichment of the Ragulator complex on Cystinosin-positive lysosomes. Together, this work uncovers lysosomal proteome heterogeneity across multiple scales and provides resources for understanding the contribution of lysosomal protein dynamics to signal transduction, organelle crosstalk, and organism longevity.

Membrane anchoring of the DIRAS3 N-terminal extension permits tumor suppressor function.

DIRAS3 is an imprinted tumor suppressor gene encoding a GTPase that has a distinctive N-terminal extension (NTE) not found in other RAS proteins. This NTE and the prenylated C-terminus are required for DIRAS3-mediated inhibition of RAS/MAP signaling and PI3K activity at the plasma membrane. In this study, we applied biochemical, biophysical, and computational methods to characterize the structure and function of the NTE. The NTE peptide recognizes phosphoinositides PI(3,4,5)P3 and PI(4,5)P2 with rapid kinetics and strong affinity. Lipid binding induces NTE structural change from disorder to amphipathic helix. Mass spectrometry identified N-myristoylation of DIRAS3. All-atom molecular dynamic simulations predict DIRAS3 could adhere to the membrane through both termini, suggesting the NTE is involved in targeting and stabilizing DIRAS3 on the membrane by double anchoring. Overall, our results are consistent with DIRAS3's function as a tumor suppressor, whereby the membrane-bound DIRAS3 can effectively target PI3K and KRAS at the membrane.

Speg interactions that regulate the stability of excitation-contraction coupling protein complexes in triads and dyads.

Here we show that striated muscle preferentially expressed protein kinase α (Spegα) maintains cardiac function in hearts with Spegß deficiency. Speg is required for stability of excitation-contraction coupling (ECC) complexes and interacts with esterase D (Esd), Cardiomyopathy-Associated Protein 5 (Cmya5), and Fibronectin Type III and SPRY Domain Containing 2 (Fsd2) in cardiac and skeletal muscle. Mice with a sequence encoding a V5/HA tag inserted into the first exon of the Speg gene (HA-Speg mice) display a >90% decrease in Spegß but Spegα is expressed at ~50% of normal levels. Mice deficient in both Spegα and Speg ß (Speg KO mice) develop a severe dilated cardiomyopathy and muscle weakness and atrophy, but HA-Speg mice display mild muscle weakness with no cardiac involvement. Spegα in HA-Speg mice suppresses Ca2+ leak, proteolytic cleavage of Jph2, and disruption of transverse tubules. Despite it's low levels, HA-Spegß immunoprecipitation identified Esd, Cmya5 and Fsd2 as Spegß binding partners that localize to triads and dyads to stabilize ECC complexes. This study suggests that Spegα and Spegß display functional redundancy, identifies Esd, Cmya5 and Fsd2 as components of both cardiac dyads and skeletal muscle triads and lays the groundwork for the identification of new therapeutic targets for centronuclear myopathy.

Daam2 phosphorylation by CK2α negatively regulates Wnt activity during white matter development and injury.

Wnt signaling plays an essential role in developmental and regenerative myelination in the central nervous system. The Wnt signaling pathway is composed of multiple regulatory layers; thus, how these processes are coordinated to orchestrate oligodendrocyte (OL) development remains unclear. Here, we show CK2α, a Wnt/ß-catenin signaling Ser/Thr kinase, phosphorylates Daam2, inhibiting its function and Wnt activity during OL development. Intriguingly, we found Daam2 phosphorylation differentially impacts distinct stages of OL development, accelerating early differentiation followed by decelerating maturation and myelination. Application toward white matter injury revealed CK2α-mediated Daam2 phosphorylation plays a protective role for developmental and behavioral recovery after neonatal hypoxia, while promoting myelin repair following adult demyelination. Together, our findings identify a unique regulatory node in the Wnt pathway that regulates OL development via protein phosphorylation-induced signaling complex instability and highlights a new biological mechanism for myelin restoration.

Identifying the Reactive Metabolites of Tyrosine Kinase Inhibitor Pexidartinib In Vitro Using LC-MS-Based Metabolomic Approaches.

Pexidartinib (PEX, TURALIO), a selective and potent inhibitor of the macrophage colony-stimulating factor-1 receptor, has been approved for the treatment of tenosynovial giant cell tumor. However, frequent and severe adverse effects have been reported in the clinic, resulting in a boxed warning on PEX for its risk of liver injury. The mechanisms underlying PEX-related hepatotoxicity, particularly metabolism-related toxicity, remain unknown. In the current study, the metabolic activation of PEX was investigated in human/mouse liver microsomes (HLM/MLM) and primary human hepatocytes (PHH) using glutathione (GSH) and methoxyamine (NH2OMe) as trapping reagents. A total of 11 PEX-GSH and 7 PEX-NH2OMe adducts were identified in HLM/MLM using an LC-MS-based metabolomics approach. Additionally, 4 PEX-GSH adducts were detected in the PHH. CYP3A4 and CYP3A5 were identified as the primary enzymes responsible for the formation of these adducts using recombinant human P450s and CYP3A chemical inhibitor ketoconazole. Overall, our studies suggested that PEX metabolism can produce reactive metabolites mediated by CYP3A, and the association of the reactive metabolites with PEX hepatotoxicity needs to be further studied.

Purification and Characterization of Mitochondrial Mg2+-Independent Sphingomyelinase from Rat Brain.

Sphingomyelinase (SMase) catalyzes ceramide production from sphingomyelin. Ceramides are critical in cellular responses such as apoptosis. They enhance mitochondrial outer membrane permeabilization (MOMP) through self-assembly in the mitochondrial outer membrane to form channels that release cytochrome c from intermembrane space (IMS) into the cytosol, triggering caspase-9 activation. However, the SMase involved in MOMP is yet to be identified. Here, we identified a mitochondrial Mg2+-independent SMase (mt-iSMase) from rat brain, which was purified 6,130-fold using a Percoll gradient, pulled down with biotinylated sphingomyelin, and subjected to Mono Q anion exchange. A single peak of mt-iSMase activity was eluted at a molecular mass of approximately 65 kDa using Superose 6 gel filtration. The purified enzyme showed optimal activity at pH of 6.5 and was inhibited by dithiothreitol and Mg2+, Mn2+, N2+, Cu2+, Zn2+, Fe2+, and Fe3+ ions. It was also inhibited by GW4869, which is a non-competitive inhibitor of Mg2+-dependent neutral SMase 2 (encoded by SMPD3), that protects against cytochrome c release-mediated cell death. Subfractionation experiments showed that mt-iSMase localizes in the IMS of the mitochondria, implying that mt-iSMase may play a critical role in generating ceramides for MOMP, cytochrome c release, and apoptosis. These data suggest that the purified enzyme in this study is a novel SMase.

Functional characterization of age-dependent p16 epimutation reveals biological drivers and therapeutic targets for colorectal cancer.

BACKGROUND: Methylation of the p16 promoter resulting in epigenetic gene silencing-known as p16 epimutation-is frequently found in human colorectal cancer and is also common in normal-appearing colonic mucosa of aging individuals. Thus, to improve clinical care of colorectal cancer (CRC) patients, we explored the role of age-related p16 epimutation in intestinal tumorigenesis. METHODS: We established a mouse model that replicates two common genetic and epigenetic events observed in human CRCs: Apc mutation and p16 epimutation. We conducted long-term survival and histological analysis of tumor development and progression. Colonic epithelial cells and tumors were collected from mice and analyzed by RNA sequencing (RNA-seq), quantitative PCR, and flow cytometry. We performed single-cell RNA sequencing (scRNA-seq) to characterize tumor-infiltrating immune cells throughout tumor progression. We tested whether anti-PD-L1 immunotherapy affects overall survival of tumor-bearing mice and whether inhibition of both epigenetic regulation and immune checkpoint is more efficacious. RESULTS: Mice carrying combined Apc mutation and p16 epimutation had significantly shortened survival and increased tumor growth compared to those with Apc mutation only. Intriguingly, colon tumors with p16 epimutation exhibited an activated interferon pathway, increased expression of programmed death-ligand 1 (Pdl1), and enhanced infiltration of immune cells. scRNA-seq further revealed the presence of Foxp3+ Tregs and γδT17 cells, which contribute to an immunosuppressive tumor microenvironment (TME). Furthermore, we showed that a combined therapy using an inhibitor of DNA methylation and a PD-L1 immune checkpoint inhibitor is more effective for improving survival in tumor-bearing mice than blockade of either pathway alone. CONCLUSIONS: Our study demonstrated that age-dependent p16 epimutation creates a permissive microenvironment for malignant transformation of polyps to colon cancer. Our findings provide a mechanistic rationale for future targeted therapy in patients with p16 epimutation.

3-Phosphoinositide-dependent kinase 1 drives acquired resistance to osimertinib.

Osimertinib sensitive and resistant NSCLC NCI-H1975 clones are used to model osimertinib acquired resistance in humanized and non-humanized mice and delineate potential resistance mechanisms. No new EGFR mutations or loss of the EGFR T790M mutation are found in resistant clones. Resistant tumors grown under continuous osimertinib pressure both in humanized and non-humanized mice show aggressive tumor regrowth which is significantly less sensitive to osimertinib as compared with parental tumors. 3-phosphoinositide-dependent kinase 1 (PDK1) is identified as a potential driver of osimertinib acquired resistance, and its selective inhibition by BX795 and CRISPR gene knock out, sensitizes resistant clones. In-vivo inhibition of PDK1 enhances the osimertinib sensitivity against osimertinib resistant xenograft and a patient derived xenograft (PDX) tumors. PDK1 knock-out dysregulates PI3K/Akt/mTOR signaling, promotes cell cycle arrest at the G1 phase. Yes-associated protein (YAP) and active-YAP are upregulated in resistant tumors, and PDK1 knock-out inhibits nuclear translocation of YAP. Higher expression of PDK1 and an association between PDK1 and YAP are found in patients with progressive disease following osimertinib treatment. PDK1 is a central upstream regulator of two critical drug resistance pathways: PI3K/AKT/mTOR and YAP.

The cancer testis antigen TDRD1 regulates prostate cancer proliferation by associating with the snRNP biogenesis machinery.

Prostate cancer is the most commonly diagnosed noncutaneous cancer in American men. TDRD1, a germ cell-specific gene, is erroneously expressed in more than half of prostate tumors, but its role in prostate cancer development remains elusive. In this study, we identified a PRMT5-TDRD1 signaling axis that regulates the proliferation of prostate cancer cells. PRMT5 is a protein arginine methyltransferase essential for small nuclear ribonucleoprotein (snRNP) biogenesis. Methylation of Sm proteins by PRMT5 is a critical initiation step for assembling snRNPs in the cytoplasm, and the final snRNP assembly takes place in Cajal bodies in the nucleus. By mass spectrum analysis, we found that TDRD1 interacts with multiple subunits of the snRNP biogenesis machinery. In the cytoplasm, TDRD1 interacts with methylated Sm proteins in a PRMT5-dependent manner. In the nucleus, TDRD1 interacts with Coilin, the scaffold protein of Cajal bodies. Ablation of TDRD1 in prostate cancer cells disrupted the integrity of Cajal bodies, affected the snRNP biogenesis, and reduced cell proliferation. Taken together, this study represents the first characterization of TDRD1 functions in prostate cancer development and suggests TDRD1 as a potential therapeutic target for prostate cancer treatment.

Astrocytic Slc4a4 regulates blood-brain barrier integrity in healthy and stroke brains via a NO-CCL2-CCR2 pathway.

Astrocytes play vital roles in blood-brain barrier (BBB) maintenance, yet how they support BBB integrity under normal or pathological conditions remains poorly defined. Recent evidence suggests pH homeostasis is a new cellular mechanism important for BBB integrity. In the current study, we investigated the function of an astrocyte-specific pH regulator, Slc4a4, in BBB maintenance and repair. We show that astrocytic Slc4a4 is required for normal astrocyte morphological complexity and BBB function. Multi-omics analyses identified increased astrocytic secretion of CCL2 coupled with dysregulated arginine-NO metabolism after Slc4a4 deletion. Using a model of ischemic stroke, we found that loss of Slc4a4 exacerbates BBB disruption and reactive gliosis, which were both rescued by pharmacological or genetic inhibition of the NO-CCL2 pathway in vivo. Together, our study identifies the astrocytic Slc4a4-NO-CCL2 axis as a pivotal mechanism controlling BBB integrity and repair, while providing insights for a novel therapeutic approach against BBB-related CNS disorders.

The cancer testis antigen TDRD1 regulates prostate cancer proliferation by associating with snRNP biogenesis machinery.

Prostate cancer is the most commonly diagnosed noncutaneous cancer in American men. TDRD1, a germ cell-specific gene, is erroneously expressed in more than half of prostate tumors, but its role in prostate cancer development remains elusive. In this study, we identified a PRMT5-TDRD1 signaling axis that regulates the proliferation of prostate cancer cells. PRMT5 is a protein arginine methyltransferase essential for small nuclear ribonucleoprotein (snRNP) biogenesis. Methylation of Sm proteins by PRMT5 is a critical initiation step for assembling snRNPs in the cytoplasm, and the final snRNP assembly takes place in Cajal bodies in the nucleus. By mass spectrum analysis, we found that TDRD1 interacts with multiple subunits of the snRNP biogenesis machinery. In the cytoplasm, TDRD1 interacts with methylated Sm proteins in a PRMT5-dependent manner. In the nucleus, TDRD1 interacts with Coilin, the scaffold protein of Cajal bodies. Ablation of TDRD1 in prostate cancer cells disrupted the integrity of Cajal bodies, affected the snRNP biogenesis, and reduced cell proliferation. Taken together, this study represents the first characterization of TDRD1 functions in prostate cancer development and suggests TDRD1 as a potential therapeutic target for prostate cancer treatment.

In Vitro Anti-Inflammatory and Antioxidant Activities of pH-Responsive Resveratrol-Urocanic Acid Nano-Assemblies.

Inflammatory environments provide vital biochemical stimuli (i.e., oxidative stress, pH, and enzymes) for triggered drug delivery in a controlled manner. Inflammation alters the local pH within the affected tissues. As a result, pH-sensitive nanomaterials can be used to effectively target drugs to the site of inflammation. Herein, we designed pH-sensitive nanoparticles in which resveratrol (an anti-inflammatory and antioxidant compound (RES)) and urocanic acid (UA) were complexed with a pH-sensitive moiety using an emulsion method. These RES-UA NPs were characterized by transmission electron microscopy, dynamic light scattering, zeta potential, and FT-IR spectroscopy. The anti-inflammatory and antioxidant activities of the RES-UA NPs were assessed in RAW 264.7 macrophages. The NPs were circular in shape and ranged in size from 106 to 180 nm. The RES-UA NPs suppressed the mRNA expression of the pro-inflammatory molecules inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages in a concentration-dependent manner. Incubation of LPS-stimulated macrophages with RES-UA NPs reduced the generation of reactive oxygen species (ROS) in a concentration-dependent manner. These results suggest that pH-responsive RES-UA NPs can be used to decrease ROS generation and inflammation.

Dawn-to-dusk dry fasting induces anti-atherosclerotic, anti-inflammatory, and anti-tumorigenic proteome in peripheral blood mononuclear cells in subjects with metabolic syndrome.

Background: Metabolic syndrome characterized by abdominal obesity, high blood pressure, elevated fasting glucose and triglyceride levels and low high-density lipoprotein cholesterol level is associated with pro-inflammatory state, increased risk for atherosclerosis, and multiple cancers. Our previous results on subjects with metabolic syndrome showed that 4-week dawn-to-dusk (sunset) dry fasting resulted in significant changes in the serum proteome and improvement in several metabolic risk factors. Peripheral blood mononuclear cells (PBMC) proteomics is a powerful tool that can provide mechanistic insights into how dawn-to-dusk dry fasting affects protein expression in metabolic pathways at cellular level. In this study, we determined whether dawn-to-dusk dry fasting would induce favorable changes in PBMC proteome in subjects with metabolic syndrome, similar to the changes induced by dawn-to-dusk dry fasting in the same subjects' serum proteome. Methods: We conducted a prospective study on subjects with metabolic syndrome and collected blood specimens before 4-week dawn-to-dusk dry fasting, at the end of 4-week dawn-to-dusk dry fasting, and one week after 4-week dawn-to-dusk dry fasting. We performed untargeted proteomics using nano ultra-high performance liquid chromatography-tandem mass spectrometry to assess the impact of 4-week dawn-to-dusk dry fasting on PBMC proteome. Results: There were 14 subjects with metabolic syndrome with a mean age of 59 who fasted from dawn to dusk (strict dry fasting without any liquid or food intake) for more than 14 h daily for 29 days. The quantitative proteome analysis showed that apolipoprotein B (APOB) gene protein products (GP) levels were downregulated and had the most statistical significance of the observed difference at the end of 4-week dawn-to-dusk dry fasting (P = 0.008) and one week after 4-week dawn-to-dusk dry fasting (P = 0.0004) compared with the levels before 4-week dawn-to-dusk dry fasting. The comparison between GP levels before and at the end of 4-week dawn-to-dusk dry fasting showed an alteration in the expression of genes associated with lipid and atherosclerosis pathway (P = 6.014e-4) and C-type lectin receptor signaling pathway (P = 1.064e-5). The genes that were differentially expressed in the lipid and atherosclerosis pathway were APOB (P = 0.008), CD36 (P = 0.040), CALM1, CALM2, CALM3 (P = 0.015), and HSPA8 (P = 0.047). One of the differentially expressed genes in the C-type lectin receptor signaling pathway was lymphocyte-specific protein 1 (LSP1), which showed an average of 19-fold increase at the end of 4-week dawn-to-dusk dry fasting compared with the GP levels before fasting (P = 0.004). Several GPs associated with tumor-suppressor effect (TUBB4B, LSP1, ACTR3B) were upregulated, and GPs associated with tumor-promoter effect (CD36, CALM1, CALM2, CALM3, FLOT2, PPIF) were downregulated at the end of 4-week dawn-to-dusk dry fasting or one week after 4-week dawn-to-dusk dry fasting compared with the GP levels before 4-week dawn-to-dusk dry fasting. Conclusion: Based on our results, we conclude that in subjects with metabolic syndrome, 4-week dawn-to-dusk dry fasting induced anti-atherosclerotic, anti-inflammatory, and anti-tumorigenic PMBC proteome. Randomized, controlled clinical trials are needed to further investigate the effect of dawn-to-dusk dry fasting on subjects with chronic metabolic diseases and metabolic syndrome-induced cancers.

ERK Inhibitor Ulixertinib Inhibits High-Risk Neuroblastoma Growth In Vitro and In Vivo.

Neuroblastoma (NB) is a pediatric tumor of the peripheral nervous system. Approximately 80% of relapsed NB show RAS-MAPK pathway mutations that activate ERK, resulting in the promotion of cell proliferation and drug resistance. Ulixertinib, a first-in-class ERK-specific inhibitor, has shown promising antitumor activity in phase 1 clinical trials for advanced solid tumors. Here, we show that ulixertinib significantly and dose-dependently inhibits cell proliferation and colony formation in different NB cell lines, including PDX cells. Transcriptomic analysis revealed that ulixertinib extensively inhibits different oncogenic and neuronal developmental pathways, including EGFR, VEGF, WNT, MAPK, NGF, and NTRK1. The proteomic analysis further revealed that ulixertinib inhibits the cell cycle and promotes apoptosis in NB cells. Additionally, ulixertinib treatment significantly sensitized NB cells to the conventional chemotherapeutic agent doxorubicin. Furthermore, ulixertinib potently inhibited NB tumor growth and prolonged the overall survival of the treated mice in two different NB mice models. Our preclinical study demonstrates that ulixertinib, either as a single agent or in combination with current therapies, is a novel and practical therapeutic approach for NB.

Modulation of the extracellular matrix by Streptococcus gallolyticus subsp. gallolyticus and importance in cell proliferation.

Streptococcus gallolyticus subspecies gallolyticus (Sgg) has a strong clinical association with colorectal cancer (CRC) and actively promotes the development of colon tumors. Previous work showed that this organism stimulates CRC cells proliferation and tumor growth. However, the molecular mechanisms underlying these activities are not well understood. Here, we found that Sgg upregulates the expression of several type of collagens in HT29 and HCT116 cells, with type VI collagen (ColVI) being the highest upregulated type. Knockdown of ColVI abolished the ability of Sgg to induce cell proliferation and reduced the adherence of Sgg to CRC cells. The extracellular matrix (ECM) is an important regulator of cell proliferation. Therefore, we further examined the role of decellularized matrix (dc-matrix), which is free of live bacteria or cells, in Sgg-induced cell proliferation. Dc-matrix prepared from Sgg-treated cells showed a significantly higher pro-proliferative activity than that from untreated cells or cells treated with control bacteria. On the other hand, dc-matrix from Sgg-treated ColVI knockdown cells showed no difference in the capacity to support cell proliferation compared to that from untreated ColVI knockdown cells, suggesting that the ECM by itself is a mediator of Sgg-induced cell proliferation. Furthermore, Sgg treatment of CRC cells but not ColVI knockdown CRC cells resulted in significantly larger tumors in vivo, suggesting that ColVI is important for Sgg to promote tumor growth in vivo. These results highlight a dynamic bidirectional interplay between Sgg and the ECM, where Sgg upregulates collagen expression. The Sgg-modified ECM in turn affects the ability of Sgg to adhere to host cells and more importantly, acts as a mediator for Sgg-induced CRC cell proliferation. Taken together, our results reveal a novel mechanism in which Sgg stimulates CRC proliferation through modulation of the ECM.

Uridine Diphosphate Glucuronosyl Transferase 2B28 (UGT2B28) Promotes Tumor Progression and Is Elevated in African American Prostate Cancer Patients.

Prostate cancer (PCa) is the second most diagnosed cancer in the United States and is associated with metabolic reprogramming and significant disparities in clinical outcomes among African American (AA) men. While the cause is likely multi-factorial, the precise reasons for this are unknown. Here, we identified a higher expression of the metabolic enzyme UGT2B28 in localized PCa and metastatic disease compared to benign adjacent tissue, in AA PCa compared to benign adjacent tissue, and in AA PCa compared to European American (EA) PCa. UGT2B28 was found to be regulated by both full-length androgen receptor (AR) and its splice variant, AR-v7. Genetic knockdown of UGT2B28 across multiple PCa cell lines (LNCaP, LAPC-4, and VCaP), both in androgen-replete and androgen-depleted states resulted in impaired 3D organoid formation and a significant delay in tumor take and growth rate of xenograft tumors, all of which were rescued by re-expression of UGT2B28. Taken together, our findings demonstrate a key role for the UGT2B28 gene in promoting prostate tumor growth.