ABSTRACT
Recombinant immunotoxins (RITs) are chimeric proteins composed of an Fv and a protein toxin being developed for cancer treatment. The Fv brings the toxin to the cancer cell, but most of the RITs do not reach the tumor and are removed by other organs. To identify cells responsible for RIT removal, and the pathway by which RITs reach these cells, we studied SS1P, a 63-kDa RIT that targets mesothelin-expressing tumors and has a short serum half-life. The major organs that remove RIT were identified by live mouse imaging of RIT labeled with FNIR-Z-759. Cells responsible for SS1P removal were identified by immunohistochemistry and intravital two-photon microscopy of kidneys of rats. The primary organ of SS1P removal is kidney followed by liver. In the kidney, SS1P passes through the glomerulus, is taken up by proximal tubular cells, and transferred to lysosomes. In the liver, macrophages are involved in removal. The short half-life of SS1P is due to its very rapid filtration by the kidney followed by degradation in proximal tubular cells of the kidney. In mice treated with SS1P, proximal tubular cells are damaged and albumin in the urine is increased. SS1P uptake by kidney is reduced by coadministration of l-lysine. Our data suggests that l-lysine administration to humans might prevent SS1P-mediated kidney damage, reduce albumin loss in urine, and alleviate capillary leak syndrome.
Subject(s)
Albuminuria/pathology , Antibodies, Monoclonal/pharmacokinetics , Capillary Leak Syndrome/pathology , Immunotoxins/pharmacokinetics , Kidney Tubules, Proximal/drug effects , Albuminuria/chemically induced , Albuminuria/prevention & control , Albuminuria/urine , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/toxicity , Capillary Leak Syndrome/chemically induced , Capillary Leak Syndrome/prevention & control , Capillary Leak Syndrome/urine , Disease Models, Animal , Female , Fluorescent Dyes/chemistry , Half-Life , Humans , Immunotoxins/administration & dosage , Immunotoxins/chemistry , Immunotoxins/toxicity , Intravital Microscopy , Kidney Glomerulus/metabolism , Kidney Tubules, Proximal/diagnostic imaging , Kidney Tubules, Proximal/metabolism , Kidney Tubules, Proximal/pathology , Lysine/administration & dosage , Mesothelin , Mice , Microscopy, Fluorescence , Neoplasms/drug therapy , Recombinant Proteins/administration & dosage , Recombinant Proteins/chemistry , Recombinant Proteins/pharmacokinetics , Recombinant Proteins/toxicity , Renal Elimination/drug effects , Serum Albumin/analysis , Serum Albumin/metabolism , Staining and LabelingABSTRACT
Glycosylation is a vital post-translational modification involved in a range of biological processes including protein folding, signaling, and cell-cell interactions. In 2011, a new type of O-linked glycosylation was discovered, wherein the side-chain oxygen of tyrosine is modified with a GalNAc residue (GalNAc-Tyr). At present, very little is known about GalNAc-Tyr prevalence, function, or biosynthesis. Herein, we describe the design and synthesis of a GalNAc-Tyr-derived hapten and its use in generating a GalNAc-Tyr selective monoclonal antibody. The antibody, G10C, has an unusually high affinity (app KD = 100 pM) and excellent selectivity for GalNAc-Tyr. We also obtained a crystal structure of the G10C Fab region in complex with 4-nitrophenyl-N-acetyl-α-d-galactosaminide (a small molecule mimic of GalNAc-Tyr) providing insights into the structural basis for high affinity and selectivity. Using this antibody, we discovered that GalNAc-Tyr is widely expressed in most human tissues, indicating that it is a ubiquitous and underappreciated post-translational modification. Localization to specific cell types and organ substructures within those tissues indicates that GalNAc-Tyr is likely regulated in a cell-specific manner. GalNAc-Tyr was also observed in a variety of cell lines and primary cells but was only present on the external cell surface in certain cancer cell lines, suggesting that GalNAc-Tyr localization may be altered in cancer cells. Collectively, the results shed new light on this under-studied form of glycosylation and provide access to new tools that will enable expanded biochemical and clinical investigations.
Subject(s)
Antibodies, Monoclonal , N-Acetylgalactosaminyltransferases , Antibodies, Monoclonal/metabolism , Cell Line , Glycosylation , Humans , N-Acetylgalactosaminyltransferases/metabolism , Tyrosine/metabolismABSTRACT
Mouse kidney parvovirus (MKPV), also known as murine chapparvovirus (MuCPV), is an emerging, highly infectious agent that has been isolated from laboratory and wild mouse populations. In immunocompromised mice, MKPV produces severe chronic interstitial nephropathy and renal failure within 4 to 5 months of infection. However, the course of disease, severity of histologic lesions, and viral shedding are uncertain for immunocompetent mice. We evaluated MKPV infections in CD-1 and Swiss Webster mice, 2 immunocompetent stocks of mice. MKPV-positive CD-1 mice (n = 30) were identified at approximately 8 weeks of age by fecal PCR (polymerase chain reaction) and were subsequently housed individually for clinical observation and diagnostic sampling. Cage swabs, fecal pellets, urine, and blood were evaluated by PCR at 100 and 128 days following the initial positive test, which identified that 28 of 30 were persistently infected and 24 of these were viremic at 100 days. Histologic lesions associated with MKPV in CD-1 (n = 31) and Swiss mice (n = 11) included lymphoplasmacytic tubulointerstitial nephritis with tubular degeneration. Inclusion bodies were rare; however, intralesional MKPV mRNA was consistently detected via in situ hybridization within tubular epithelial cells of the renal cortex and within collecting duct lumina. In immunocompetent CD-1 mice, MKPV infection resulted in persistent shedding of virus for up to 10 months and a mild tubulointerstitial nephritis, raising concerns that this virus could produce study variations in immunocompetent models. Intranuclear inclusions were not a consistent feature of MKPV infection in immunocompetent mice.
Subject(s)
Nephritis, Interstitial , Parvoviridae Infections , Parvovirinae , Rodent Diseases , Animals , Kidney , Mice , Mice, Inbred Strains , Nephritis, Interstitial/veterinary , Parvoviridae Infections/veterinary , Parvovirinae/pathogenicityABSTRACT
EGR1 is an early growth response zinc finger transcription factor with broad actions, including in differentiation, mitogenesis, tumor suppression, and neuronal plasticity. Here we demonstrate that Egr1-/- mice on the C57BL/6 background have normal eyelid development, but back-crossing to BALB/c background for four or five generations resulted in defective eyelid development by day E15.5, at which time EGR1 was expressed in eyelids of WT mice. Defective eyelid formation correlated with profound ocular anomalies evident by postnatal days 1-4, including severe cryptophthalmos, microphthalmia or anophthalmia, retinal dysplasia, keratitis, corneal neovascularization, cataracts, and calcification. The BALB/c albino phenotype-associated Tyrc tyrosinase mutation appeared to contribute to the phenotype, because crossing the independent Tyrc-2J allele to Egr1-/- C57BL/6 mice also produced ocular abnormalities, albeit less severe than those in Egr1-/- BALB/c mice. Thus EGR1, in a genetic background-dependent manner, plays a critical role in mammalian eyelid development and closure, with subsequent impact on ocular integrity.
Subject(s)
Eyelids/growth & development , Mice/genetics , Mice/metabolism , Animals , Early Growth Response Protein 1/genetics , Early Growth Response Protein 1/metabolism , Eye/growth & development , Eye/metabolism , Eyelids/metabolism , Gene Expression Regulation, Developmental , Mice/growth & development , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, KnockoutABSTRACT
OBJECTIVE: The goal is to evaluate avelumab, an anti-PD-L1 monoclonal immunoglobulin G antibody labeled with zirconium-89 in human PD-L1-expressing cancer cells and mouse xenografts for clinical translation. METHODS: [89Zr]Zr-DFO-PD-L1 monoclonal antibody (mAb) was synthesized using avelumab conjugated to desferrioxamine. In vitro binding studies and biodistribution studies were performed with PD-L1+MDA-MB231 cells and MDA-MB231 xenograft mouse models, respectively. Biodistributions were determined at 1, 2, 3, 5, and 7 days post coinjection of [89Zr]Zr-DFO-PD-L1 mAb without or with unlabeled avelumab (10, 20, 40, and 400 µg). RESULTS: [89Zr]Zr-DFO-PD-L1 mAb exhibited high affinity (Kd â¼ 0.3 nM) and detected moderate PD-L1 expression levels in MDA-MB231 cells. The spleen and lymph nodes exhibited the highest [89Zr]Zr-DFO-PD-L1 mAb uptakes in all time points, while MDA-MB231 tumor uptakes were lower but highly retained. In the unlabeled avelumab dose escalation studies, spleen tissue-muscle ratios decreased in a dose-dependent manner indicating specific [89Zr]Zr-DFO-PD-L1 mAb binding to PD-L1. In contrast, lymph node and tumor tissue-muscle ratios increased 4- to 5-fold at 20 and 40 µg avelumab doses. CONCLUSIONS: [89Zr]Zr-DFO-PD-L1 mAb exhibited specific and high affinity for PD-L1 in vitro and had target tissue uptakes correlating with PD-L1 expression levels in vivo. [89Zr]Zr-DFO-PD-L1 mAb uptake in PD-L1+tumors increased with escalating doses of avelumab.
Subject(s)
Antibodies, Monoclonal/administration & dosage , B7-H1 Antigen/metabolism , Breast Neoplasms/drug therapy , Deferoxamine/chemistry , Radioisotopes/chemistry , Zirconium/chemistry , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/pharmacokinetics , Antibodies, Monoclonal, Humanized , Breast Neoplasms/diagnostic imaging , Breast Neoplasms/metabolism , Dose-Response Relationship, Drug , Down-Regulation , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Immunoconjugates , Mice , Positron-Emission Tomography , Tissue Distribution , Treatment Outcome , Xenograft Model Antitumor AssaysABSTRACT
Background: Despite advances in immunotherapeutic strategies for neuroblastoma (NBL), relapse remains a significant cause of mortality for high risk patients. The discovery of novel tumor associated antigens to improve efficacy and minimize the toxicities of immunotherapy is therefore warranted. Receptor Tyrosine Kinase-like Orphan Receptor-1 and 2 (ROR1 and ROR2) have been found to be expressed in several malignancies with limited expression in healthy tissues. Objectives: Given their role in tumor migration and proliferation and the fact that they were originally cloned from a NBL cell line, we hypothesized that ROR1 and ROR2 could serve as potential targets for anti-ROR1 and anti-ROR2 based immunotherapies in NBL. Methods: We characterized the mRNA and protein expression of ROR1 and ROR2 in NBL cell lines and tissue microarrays of patient samples. To explore the potential of ROR1 targeting, we performed in vitro cytotoxicity assays against NBL using NK92 cells as effector cells. Results: Both ROR1 and ROR2 are expressed across all stages of NBL. In patients with non-MYC amplified tumors, expression of ROR1/ROR2 correlated with survival and prognosis. Moreover, in a proof-of-concept experiment, pretreatment of NBL cell line with anti-ROR1 antibody showed additive cytotoxicity with NK92 cells. Conclusions: ROR1 and ROR2 could serve as novel targets for immunotherapy in NBL. The additive effect of anti-ROR1 antibodies with NK cells needs to be explored further to evaluate the possibility of combining anti-ROR1 antibodies with immune effectors such as NK92 cells as a potential off-the shelf immunotherapy for NBL and other ROR1 expressing malignancies.
Subject(s)
Immunotherapy/methods , Neuroblastoma/immunology , Receptor Tyrosine Kinase-like Orphan Receptors/immunology , Cell Line, Tumor , Humans , PrognosisABSTRACT
Reactive oxygen species (ROS) play a critical role in cell signaling and proliferation. NADPH oxidase 1 (NOX1), a membrane-bound flavin dehydrogenase that generates O2ÌÌ, is highly expressed in colon cancer. To investigate the role that NOX1 plays in colon cancer growth, we used shRNA to decrease NOX1 expression stably in HT-29 human colon cancer cells. The 80-90% decrease in NOX1 expression achieved by RNAi produced a significant decline in ROS production and a G1/S block that translated into a 2-3-fold increase in tumor cell doubling time without increased apoptosis. The block at the G1/S checkpoint was associated with a significant decrease in cyclin D1 expression and profound inhibition of mitogen-activated protein kinase (MAPK) signaling. Decreased steady-state MAPK phosphorylation occurred concomitant with a significant increase in protein phosphatase activity for two colon cancer cell lines in which NOX1 expression was knocked down by RNAi. Diminished NOX1 expression also contributed to decreased growth, blood vessel density, and VEGF and hypoxia-inducible factor 1α (HIF-1α) expression in HT-29 xenografts initiated from NOX1 knockdown cells. Microarray analysis, supplemented by real-time PCR and Western blotting, revealed that the expression of critical regulators of cell proliferation and angiogenesis, including c-MYC, c-MYB, and VEGF, were down-regulated in association with a decline in hypoxic HIF-1α protein expression downstream of silenced NOX1 in both colon cancer cell lines and xenografts. These studies suggest a role for NOX1 in maintaining the proliferative phenotype of some colon cancers and the potential of NOX1 as a therapeutic target in this disease.
Subject(s)
Colonic Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , MAP Kinase Signaling System , NADPH Oxidases/metabolism , Reactive Oxygen Species/metabolism , Animals , Apoptosis , Cell Cycle , Cell Line, Tumor , Cell Proliferation , Colon/metabolism , Cyclin D1/metabolism , HT29 Cells , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Male , Mice , NADPH Oxidase 1 , Neoplasm Transplantation , Phenotype , Phosphorylation , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , RNA Interference , RNA, Small Interfering/metabolism , Vascular Endothelial Growth Factor A/metabolismABSTRACT
NADPH oxidase 5 (NOX5) generated reactive oxygen species (ROS) have been implicated in signaling cascades that regulate cancer cell proliferation. To evaluate and validate NOX5 expression in human tumors, we screened a broad range of tissue microarrays (TMAs), and report substantial overexpression of NOX5 in malignant melanoma and cancers of the prostate, breast, and ovary. In human UACC-257 melanoma cells that possesses high levels of functional endogenous NOX5, overexpression of NOX5 resulted in enhanced cell growth, increased numbers of BrdU positive cells, and increased γ-H2AX levels. Additionally, NOX5-overexpressing (stable and inducible) UACC-257 cells demonstrated increased normoxic HIF-1α expression and decreased p27Kip1 expression. Similarly, increased normoxic HIF-1α expression and decreased p27Kip1 expression were observed in stable NOX5-overexpressing clones of KARPAS 299 human lymphoma cells and in the human prostate cancer cell line, PC-3. Conversely, knockdown of endogenous NOX5 in UACC-257 cells resulted in decreased cell growth, decreased HIF-1α expression, and increased p27Kip1 expression. Likewise, in an additional human melanoma cell line, WM852, and in PC-3 cells, transient knockdown of endogenous NOX5 resulted in increased p27Kip1 and decreased HIF-1α expression. Knockdown of endogenous NOX5 in UACC-257 cells resulted in decreased Akt and GSK3ß phosphorylation, signaling pathways known to modulate p27Kip1 levels. In summary, our findings suggest that NOX5 expression in human UACC-257 melanoma cells could contribute to cell proliferation due, in part, to the generation of high local concentrations of extracellular ROS that modulate multiple pathways that regulate HIF-1α and networks that signal through Akt/GSK3ß/p27Kip1 .
Subject(s)
Cyclin-Dependent Kinase Inhibitor p27/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , NADPH Oxidase 5/metabolism , Reactive Oxygen Species/metabolism , Signal Transduction , Blotting, Western , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/genetics , Cyclin-Dependent Kinase Inhibitor p27/genetics , Female , Glycogen Synthase Kinase 3 beta/metabolism , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Male , Melanoma/genetics , Melanoma/metabolism , Melanoma/pathology , NADPH Oxidase 5/genetics , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Phosphorylation , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Proto-Oncogene Proteins c-akt/metabolism , RNA InterferenceABSTRACT
Increased serum levels of IL-15 are reported in type 1 diabetes (T1D). Here we report elevated serum soluble IL-15Rα levels in human T1D. To investigate the role of IL-15/IL-15Rα in the pathogenesis of T1D, we generated double transgenic mice with pancreatic ß-cell expression of IL-15 and IL-15Rα. The mice developed hyperglycemia, marked mononuclear cell infiltration, ß-cell destruction, and anti-insulin autoantibodies that mimic early human T1D. The diabetes in this model was reversed by inhibiting IL-15 signaling with anti-IL2/IL15Rß (anti-CD122), which blocks IL-15 transpresentation. Furthermore, the diabetes could be reversed by administration of the Janus kinase 2/3 inhibitor tofacitinib, which blocks IL-15 signaling. In an alternative diabetes model, nonobese diabetic mice, IL15/IL-15Rα expression was increased in islet cells in the prediabetic stage, and inhibition of IL-15 signaling with anti-CD122 at the prediabetic stage delayed diabetes development. In support of the view that these observations reflect the conditions in humans, we demonstrated pancreatic islet expression of both IL-15 and IL-15Rα in human T1D. Taken together our data suggest that disordered IL-15 and IL-15Rα may be involved in T1D pathogenesis and the IL-15/IL15Rα system and its signaling pathway may be rational therapeutic targets for early T1D.
Subject(s)
Diabetes Mellitus, Type 1/etiology , Disease Models, Animal , Insulin-Secreting Cells/metabolism , Interleukin-15 Receptor alpha Subunit/metabolism , Interleukin-15/metabolism , Signal Transduction/drug effects , Animals , Humans , Interleukin-15/antagonists & inhibitors , Interleukin-15/blood , Interleukin-15 Receptor alpha Subunit/blood , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, Transgenic , Piperidines/pharmacology , Pyrimidines/pharmacology , Pyrroles/pharmacologyABSTRACT
Pancreatitis is associated with release of proinflammatory cytokines and reactive oxygen species and plays an important role in the development of pancreatic cancer. We recently demonstrated that dual oxidase (Duox)2, an NADPH oxidase essential for reactive oxygen species-related, gastrointestinal host defense, is regulated by IFN-γ-mediated Stat1 binding to the Duox2 promoter in pancreatic tumor lines. Because LPS enhances the development and invasiveness of pancreatic cancer in vivo following TLR4-related activation of NF-κB, we examined whether LPS, alone or combined with IFN-γ, regulated Duox2. We found that upregulation of TLR4 by IFN-γ in BxPC-3 and CFPAC-1 pancreatic cancer cells was augmented by LPS, resulting in activation of NF-κB, accumulation of NF-κB (p65) in the nucleus, and increased binding of p65 to the Duox2 promoter. TLR4 silencing with small interfering RNAs, as well as two independent NF-κB inhibitors, attenuated LPS- and IFN-γ-mediated Duox2 upregulation in BxPC-3 cells. Induction of Duox2 expression by IFN-γ and LPS may result from IFN-γ-related activation of Stat1 acting in concert with NF-κB-related upregulation of Duox2. Sustained extracellular accumulation of H(2)O(2) generated by exposure to both LPS and IFN-γ was responsible for an â¼50% decrease in BxPC-3 cell proliferation associated with a G(1) cell cycle block, apoptosis, and DNA damage. We also demonstrated upregulation of Duox expression in vivo in pancreatic cancer xenografts and in patients with chronic pancreatitis. These results suggest that inflammatory cytokines can interact to produce a Duox-dependent pro-oxidant milieu that could increase the pathologic potential of pancreatic inflammation and pancreatic cancer cells.
Subject(s)
Interferon-gamma/physiology , Lipopolysaccharides/physiology , Membrane Proteins/biosynthesis , NADPH Oxidases/biosynthesis , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/metabolism , Toll-Like Receptor 4/metabolism , Animals , Cell Line, Tumor , Chronic Disease , Dual Oxidases , Female , Humans , Mice , Mice, Nude , Neoplasm Transplantation , Pancreatic Neoplasms/enzymology , Pancreatitis/enzymology , Pancreatitis/immunology , Pancreatitis/metabolism , Random Allocation , Reactive Oxygen Species/metabolism , Signal Transduction/immunology , Toll-Like Receptor 4/physiology , Tumor Cells, CulturedABSTRACT
A primary issue with nanomedicine biological evaluation is determination of nanoparticle carrier tissue distribution and stability. Here we present a method to evaluate nanomedicine distribution in tissues that is applicable to most nanomedicine constructs. This method utilizes immunohistochemical (IHC) analysis of an Alexa Fluor 488-tag and/or polyethylene glycol (PEG), a very common nanomedicine component, for tissue localization. Using specific Alexa Fluor 488- and/or PEG antibody-based IHC staining procedures allows evaluation of high-resolution nanoparticle tissue distribution, nanoparticle tissue stability, and also allows correlation of distribution with morphological changes. This protocol outlines the methods to follow to ensure proper tissue collection and optimized immunohistochemical staining of Alexa Fluor 488-tag and PEG in tissues.
Subject(s)
Fluoresceins , Fluorescent Dyes , Polyethylene Glycols , Sulfonic Acids , Immunohistochemistry , Nanomedicine , Tissue DistributionABSTRACT
Background: A principal protective component of the mammalian blood-brain barrier (BBB) is the high expression of the multidrug efflux transporters P-glycoprotein (P-gp, encoded by ABCB1) and ABCG2 (encoded by ABCG2) on the lumenal surface of endothelial cells. The zebrafish P-gp homolog Abcb4 is expressed at the BBB and phenocopies human P-gp. Comparatively little is known about the four zebrafish homologs of the human ABCG2 gene: abcg2a, abcg2b, abcg2c, and abcg2d. Here we report the functional characterization and brain tissue distribution of zebrafish ABCG2 homologs. Methods: To determine substrates of the transporters, we stably expressed each in HEK-293 cells and performed cytotoxicity and fluorescent efflux assays with known ABCG2 substrates. To assess the expression of transporter homologs, we used a combination of RNAscope in situ hybridization probes and immunohistochemistry to stain paraffin-embedded sections of adult and larval zebrafish. Results: We found Abcg2a had the greatest substrate overlap with ABCG2, and Abcg2d appeared to be the least functionally similar. We identified abcg2a as the only homolog expressed at the adult and larval zebrafish BBB, based on its localization to claudin-5 positive brain vasculature. Conclusions: These results demonstrate the conserved function of zebrafish Abcg2a and suggest that zebrafish may be an appropriate model organism for the studying the role of ABCG2 at the BBB.
ABSTRACT
BACKGROUND: A principal protective component of the mammalian blood-brain barrier (BBB) is the high expression of the multidrug efflux transporters P-glycoprotein (P-gp, encoded by ABCB1) and ABCG2 (encoded by ABCG2) on the lumenal surface of endothelial cells. The zebrafish P-gp homolog Abcb4 is expressed at the BBB and phenocopies human P-gp. Comparatively little is known about the four zebrafish homologs of the human ABCG2 gene: abcg2a, abcg2b, abcg2c, and abcg2d. Here we report the functional characterization and brain tissue distribution of zebrafish ABCG2 homologs. METHODS: To determine substrates of the transporters, we stably expressed each in HEK-293 cells and performed cytotoxicity and fluorescent efflux assays with known ABCG2 substrates. To assess the expression of transporter homologs, we used a combination of RNAscope in situ hybridization probes and immunohistochemistry to stain paraffin-embedded sections of adult and larval zebrafish. RESULTS: We found Abcg2a had the greatest substrate overlap with ABCG2, and Abcg2d appeared to be the least functionally similar. We identified abcg2a as the only homolog expressed at the adult and larval zebrafish BBB, based on its localization to claudin-5 positive brain vasculature. CONCLUSIONS: These results demonstrate the conserved function of zebrafish Abcg2a and suggest that zebrafish may be an appropriate model organism for studying the role of ABCG2 at the BBB.
Subject(s)
ATP Binding Cassette Transporter, Subfamily G, Member 2 , Blood-Brain Barrier , Zebrafish , Adult , Animals , Humans , ATP Binding Cassette Transporter, Subfamily G, Member 2/genetics , ATP Binding Cassette Transporter, Subfamily G, Member 2/metabolism , Blood-Brain Barrier/metabolism , Endothelial Cells/metabolism , HEK293 Cells , Mammals/metabolism , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Zebrafish/metabolismABSTRACT
In malignant glioma, cytotoxic drugs are often inhibited from accessing the tumor site due to the blood-tumor barrier (BTB). Ibrutinib, FDA-approved lymphoma agent, inhibits Bruton tyrosine kinase (BTK) and has previously been shown to independently impair aortic endothelial adhesion and increase rodent glioma model survival in combination with cytotoxic therapy. Yet additional research is required to understand ibrutinib's effect on BTB function. In this study, we detail baseline BTK expression in glioma cells and its surrounding vasculature, then measure endothelial junctional expression/function changes with varied ibrutinib doses in vitro. Rat glioma cells and rodent glioma models were treated with ibrutinib alone (1-10 µM and 25 mg/kg) and in combination with doxil (10-100 µM and 3 mg/kg) to assess additive effects on viability, drug concentrations, tumor volume, endothelial junctional expression and survival. We found that ibrutinib, in a dose-dependent manner, decreased brain endothelial cell-cell adhesion over 24 h, without affecting endothelial cell viability (p < 0.005). Expression of tight junction gene and protein expression was decreased maximally 4 h after administration, along with inhibition of efflux transporter, ABCB1, activity. We demonstrated an additive effect of ibrutinib with doxil on rat glioma cells, as seen by a significant reduction in cell viability (p < 0.001) and increased CNS doxil concentration in the brain (56 ng/mL doxil alone vs. 74.6 ng/mL combination, p < 0.05). Finally, Ibrutinib, combined with doxil, prolonged median survival in rodent glioma models (27 vs. 16 days, p < 0.0001) with brain imaging showing a - 53% versus - 75% volume change with doxil alone versus combination therapy (p < 0.05). These findings indicate ibrutinib's ability to increase brain endothelial permeability via junctional disruption and efflux inhibition, to increase BTB drug entry and prolong rodent glioma model survival. Our results motivate the need to identify other BTB modifiers, all with the intent of improving survival and reducing systemic toxicities.
Subject(s)
Adenine/analogs & derivatives , Antineoplastic Agents , Doxorubicin/analogs & derivatives , Glioma , Piperidines , Rats , Animals , Rodentia , Glioma/pathology , Antineoplastic Agents/therapeutic use , Blood-Brain Barrier/pathology , Polyethylene GlycolsABSTRACT
Immune therapy is the new frontier of cancer treatment. Therapeutic radiation is a known inducer of immune response and can be limited by immunosuppressive mediators including cyclooxygenase-2 (COX2) that is highly expressed in aggressive triple negative breast cancer (TNBC). A clinical cohort of TNBC tumors revealed poor radiation therapeutic efficacy in tumors expressing high COX2. Herein, we show that radiation combined with adjuvant NSAID (indomethacin) treatment provides a powerful combination to reduce both primary tumor growth and lung metastasis in aggressive 4T1 TNBC tumors, which occurs in part through increased antitumor immune response. Spatial immunological changes including augmented lymphoid infiltration into the tumor epithelium and locally increased cGAS/STING1 and type I IFN gene expression were observed in radiation-indomethacin-treated 4T1 tumors. Thus, radiation and adjuvant NSAID treatment shifts "immune desert phenotypes" toward antitumor M1/TH1 immune mediators in these immunologically challenging tumors. Importantly, radiation-indomethacin combination treatment improved local control of the primary lesion, reduced metastatic burden, and increased median survival when compared with radiation treatment alone. These results show that clinically available NSAIDs can improve radiation therapeutic efficacy through increased antitumor immune response and augmented local generation of cGAS/STING1 and type I IFNs.
Subject(s)
Membrane Proteins , Signal Transduction , T-Lymphocytes, Cytotoxic , Animals , Membrane Proteins/metabolism , Mice , Female , Signal Transduction/drug effects , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/drug effects , Triple Negative Breast Neoplasms/pathology , Triple Negative Breast Neoplasms/immunology , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/radiotherapy , Indomethacin/pharmacology , Indomethacin/therapeutic use , Cell Line, Tumor , Humans , Lung Neoplasms/immunology , Lung Neoplasms/pathology , Lung Neoplasms/drug therapy , Cyclooxygenase Inhibitors/pharmacology , Cyclooxygenase Inhibitors/therapeutic use , Nucleotidyltransferases/metabolism , Interferon Type I/metabolism , Cyclooxygenase 2/metabolism , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/drug effects , Mice, Inbred BALB CABSTRACT
Histone deacetylase inhibitors (HDACi) are part of a growing class of epigenetic therapies used for the treatment of cancer. Although HDACis are effective in the treatment of T-cell lymphomas, treatment of solid tumors with this class of drugs has not been successful. Overexpression of the multidrug resistance protein P-glycoprotein (P-gp), encoded by ABCB1, is known to confer resistance to the HDACi romidepsin in vitro, yet increased ABCB1 expression has not been associated with resistance in patients, suggesting that other mechanisms of resistance arise in the clinic. To identify alternative mechanisms of resistance to romidepsin, we selected MCF-7 breast cancer cells with romidepsin in the presence of the P-gp inhibitor verapamil to reduce the likelihood of P-gp-mediated resistance. The resulting cell line, MCF-7 DpVp300, does not express P-gp and was found to be selectively resistant to romidepsin but not to other HDACis such as belinostat, panobinostat, or vorinostat. RNA-sequencing analysis revealed upregulation of the mRNA coding for the putative methyltransferase, METTL7A, whose paralog, METTL7B, was previously shown to methylate thiol groups on hydrogen sulfide and captopril. As romidepsin has a thiol as the zinc-binding moiety, we hypothesized that METTL7A could inactivate romidepsin and other thiol-based HDACis via methylation of the thiol group. We demonstrate that expression of METTL7A or METTL7B confers resistance to thiol-based HDACis and that both enzymes are capable of methylating thiol-containing HDACis. We thus propose that METTL7A and METTL7B confer resistance to thiol-based HDACis by methylating and inactivating the zinc-binding thiol.
Subject(s)
Histone Deacetylase Inhibitors , Neoplasms , Humans , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylase Inhibitors/therapeutic use , Methyltransferases/metabolism , Neoplasms/drug therapy , Panobinostat/pharmacology , Panobinostat/therapeutic use , ZincABSTRACT
Small-cell lung cancer (SCLC) is the most fatal form of lung cancer. Intratumoral heterogeneity, marked by neuroendocrine (NE) and non-neuroendocrine (non-NE) cell states, defines SCLC, but the cell-extrinsic drivers of SCLC plasticity are poorly understood. To map the landscape of SCLC tumor microenvironment (TME), we apply spatially resolved transcriptomics and quantitative mass spectrometry-based proteomics to metastatic SCLC tumors obtained via rapid autopsy. The phenotype and overall composition of non-malignant cells in the TME exhibit substantial variability, closely mirroring the tumor phenotype, suggesting TME-driven reprogramming of NE cell states. We identify cancer-associated fibroblasts (CAFs) as a crucial element of SCLC TME heterogeneity, contributing to immune exclusion, and predicting exceptionally poor prognosis. Our work provides a comprehensive map of SCLC tumor and TME ecosystems, emphasizing their pivotal role in SCLC's adaptable nature, opening possibilities for reprogramming the TME-tumor communications that shape SCLC tumor states.
Subject(s)
Lung Neoplasms , Small Cell Lung Carcinoma , Tumor Microenvironment , Humans , Small Cell Lung Carcinoma/pathology , Small Cell Lung Carcinoma/genetics , Small Cell Lung Carcinoma/metabolism , Lung Neoplasms/pathology , Lung Neoplasms/metabolism , Cancer-Associated Fibroblasts/metabolism , Cancer-Associated Fibroblasts/pathology , Neuroendocrine Tumors/pathology , Neuroendocrine Tumors/genetics , Neuroendocrine Tumors/metabolism , Neuroendocrine Cells/pathology , Neuroendocrine Cells/metabolism , Female , Male , PrognosisABSTRACT
Thymic epithelial cells (TEC) control T cell development and play essential roles in establishing self-tolerance. By using Foxn1-Cre-driven ablation of Klf6 gene in TEC, we identified Klf6 as a critical factor in TEC development. Klf6 deficiency resulted in a hypoplastic thymus-evident from fetal stages into adulthood-in which a dramatic increase in the frequency of apoptotic TEC was observed. Among cortical TEC (cTEC), a previously unreported cTEC population expressing the transcription factor Sox10 was relatively expanded. Within medullary TEC (mTEC), mTEC I and Tuft-like mTEC IV were disproportionately decreased. Klf6 deficiency altered chromatin accessibility and affected TEC chromatin configuration. Consistent with these defects, naïve conventional T cells and invariant natural killer T cells were reduced in the spleen. Late stages of T cell receptor-dependent selection of thymocytes were affected, and mice exhibited autoimmunity. Thus, Klf6 has a prosurvival role and affects the development of specific TEC subsets contributing to thymic function.
Subject(s)
Gene Expression Regulation , Thymocytes , Animals , Mice , Cell Differentiation/genetics , Chromatin/metabolism , Epithelial Cells/metabolism , Mice, Inbred C57BL , Thymocytes/metabolism , Thymus Gland/metabolismABSTRACT
BACKGROUND: Epithelial ovarian cancer (EOC) is a global health burden, with the poorest five-year survival rate of the gynecological malignancies due to diagnosis at advanced stage and high recurrence rate. Recurrence in EOC is driven by the survival of chemoresistant, stem-like tumor-initiating cells (TICs) that are supported by a complex extracellular matrix and immunosuppressive microenvironment. To target TICs to prevent recurrence, we identified genes critical for TIC viability from a whole genome siRNA screen. A top hit was the cancer-associated, proteoglycan subunit synthesis enzyme UDP-glucose dehydrogenase (UGDH). METHODS: Immunohistochemistry was used to characterize UGDH expression in histological and molecular subtypes of EOC. EOC cell lines were subtyped according to the molecular subtypes and the functional effects of modulating UGDH expression in vitro and in vivo in C1/Mesenchymal and C4/Differentiated subtype cell lines was examined. RESULTS: High UGDH expression was observed in high-grade serous ovarian cancers and a distinctive survival prognostic for UGDH expression was revealed when serous cancers were stratified by molecular subtype. High UGDH was associated with a poor prognosis in the C1/Mesenchymal subtype and low UGDH was associated with poor prognosis in the C4/Differentiated subtype. Knockdown of UGDH in the C1/mesenchymal molecular subtype reduced spheroid formation and viability and reduced the CD133 + /ALDH high TIC population. Conversely, overexpression of UGDH in the C4/Differentiated subtype reduced the TIC population. In co-culture models, UGDH expression in spheroids affected the gene expression of mesothelial cells causing changes to matrix remodeling proteins, and fibroblast collagen production. Inflammatory cytokine expression of spheroids was altered by UGDH expression. The effect of UGDH knockdown or overexpression in the C1/ Mesenchymal and C4/Differentiated subtypes respectively was tested on mouse intrabursal xenografts and showed dynamic changes to the tumor stroma. Knockdown of UGDH improved survival and reduced tumor burden in C1/Mesenchymal compared to controls. CONCLUSIONS: These data show that modulation of UGDH expression in ovarian cancer reveals distinct roles for UGDH in the C1/Mesenchymal and C4/Differentiated molecular subtypes of EOC, influencing the tumor microenvironmental composition. UGDH is a strong potential therapeutic target in TICs, for the treatment of EOC, particularly in patients with the mesenchymal molecular subtype.
Subject(s)
Carcinoma, Ovarian Epithelial , Ovarian Neoplasms , Tumor Microenvironment , Uridine Diphosphate Glucose Dehydrogenase , Animals , Female , Humans , Mice , Carcinoma, Ovarian Epithelial/genetics , Carcinoma, Ovarian Epithelial/pathology , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Prognosis , RNA, Small Interfering/genetics , Tumor Microenvironment/genetics , Tumor Microenvironment/immunology , Uridine Diphosphate Glucose Dehydrogenase/genetics , Uridine Diphosphate Glucose Dehydrogenase/immunologyABSTRACT
PURPOSE: PAX-fusion negative rhabdomyosarcoma (FN RMS) is driven by alterations in the RAS/MAP kinase pathway and is partially responsive to MEK inhibition. Overexpression of IGF1R and its ligands is also observed in FN RMS. Preclinical and clinical studies have suggested that IGF1R is itself an important target in FN RMS. Our previous studies revealed preclinical efficacy of the MEK1/2 inhibitor, trametinib, and an IGF1R inhibitor, BMS-754807, but this combination was not pursued clinically due to intolerability in preclinical murine models. Here, we sought to identify a combination of an MEK1/2 inhibitor and IGF1R inhibitor, which would be tolerated in murine models and effective in both cell line and patient-derived xenograft models of RAS-mutant FN RMS. EXPERIMENTAL DESIGN: Using proliferation and apoptosis assays, we studied the factorial effects of trametinib and ganitumab (AMG 479), a mAb with specificity for human and murine IGF1R, in a panel of RAS-mutant FN RMS cell lines. The molecular mechanism of the observed synergy was determined using conventional and capillary immunoassays. The efficacy and tolerability of trametinib/ganitumab was assessed using a panel of RAS-mutated cell-line and patient-derived RMS xenograft models. RESULTS: Treatment with trametinib and ganitumab resulted in synergistic cellular growth inhibition in all cell lines tested and inhibition of tumor growth in four of six models of RAS-mutant RMS. The combination had little effect on body weight and did not produce thrombocytopenia, neutropenia, or hyperinsulinemia in tumor-bearing SCID beige mice. Mechanistically, ganitumab treatment prevented the phosphorylation of AKT induced by MEK inhibition alone. Therapeutic response to the combination was observed in models without a mutation in the PI3K/PTEN axis. CONCLUSIONS: We demonstrate that combined trametinib and ganitumab is effective in a genomically diverse panel of RAS-mutated FN RMS preclinical models. Our data also show that the trametinib/ganitumab combination likely has a favorable tolerability profile. These data support testing this combination in a phase I/II clinical trial for pediatric patients with relapsed or refractory RAS-mutated FN RMS.