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1.
Clin Cancer Res ; 26(11): 2711-2724, 2020 06 01.
Article in English | MEDLINE | ID: mdl-31969339

ABSTRACT

PURPOSE: Exploitation of altered glycosylation in cancer is a major goal for the design of new cancer therapy. Here, we designed a novel secreted chimeric signal peptide-Galectin-3 conjugate (sGal-3) and investigated its ability to induce cancer-specific cell death by targeting aberrantly N-glycosylated cell surface receptors on cancer cells. EXPERIMENTAL DESIGN: sGal-3 was genetically engineered from Gal-3 by extending its N-terminus with a noncleavable signal peptide from tissue plasminogen activator. sGal-3 killing ability was tested on normal and tumor cells in vitro and its antitumor activity was evaluated in subcutaneous lung cancer and orthotopic malignant glioma models. The mechanism of killing was investigated through assays detecting sGal-3 interaction with specific glycans on the surface of tumor cells and the elicited downstream proapoptotic signaling. RESULTS: We found sGal-3 preferentially binds to ß1 integrin on the surface of tumor cells due to aberrant N-glycosylation resulting from cancer-associated upregulation of several glycosyltransferases. This interaction induces potent cancer-specific death by triggering an oncoglycan-ß1/calpain/caspase-9 proapoptotic signaling cascade. sGal-3 could reduce the growth of subcutaneous lung cancers and malignant gliomas in brain, leading to increased animal survival. CONCLUSIONS: We demonstrate that sGal-3 kills aberrantly glycosylated tumor cells and antagonizes tumor growth through a novel integrin ß1-dependent cell-extrinsic apoptotic pathway. These findings provide proof-of-principle that aberrant N-oncoglycans represent valid cancer targets and support further translation of the chimeric sGal-3 peptide conjugate for cancer therapy.


Subject(s)
Apoptosis , Blood Proteins/metabolism , Galectins/metabolism , Glioma/drug therapy , Integrin beta1/metabolism , Peptide Fragments/pharmacology , Protein Sorting Signals , Animals , Blood Proteins/genetics , Cell Proliferation , Female , Galectins/genetics , Glioma/metabolism , Glioma/pathology , Glycosylation , Humans , Integrin beta1/genetics , Mice , Mice, Nude , Signal Transduction , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
2.
Nat Commun ; 10(1): 3000, 2019 07 05.
Article in English | MEDLINE | ID: mdl-31278254

ABSTRACT

Tumor-driven immune suppression is a major barrier to successful immunotherapy in ovarian carcinomas (OvCa). Among various mechanisms responsible for immune suppression, arginase-1 (ARG1)-carrying small extracellular vesicles (EVs) emerge as important contributors to tumor growth and tumor escape from the host immune system. Here, we report that small EVs found in the ascites and plasma of OvCa patients contain ARG1. EVs suppress proliferation of CD4+ and CD8+ T-cells in vitro and in vivo in OvCa mouse models. In mice, ARG1-containing EVs are transported to draining lymph nodes, taken up by dendritic cells and inhibit antigen-specific T-cell proliferation. Increased expression of ARG1 in mouse OvCa cells is associated with accelerated tumor progression that can be blocked by an arginase inhibitor. Altogether, our studies show that tumor cells use EVs as vehicles to carry over long distances and deliver to immune cells a metabolic checkpoint molecule - ARG1, mitigating anti-tumor immune responses.


Subject(s)
Arginase/metabolism , Extracellular Vesicles/immunology , Ovarian Neoplasms/immunology , Tumor Escape/immunology , Animals , Arginase/antagonists & inhibitors , Arginase/immunology , Ascites/immunology , Ascites/pathology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Communication/immunology , Cell Line, Tumor/transplantation , Cell Proliferation/drug effects , Cohort Studies , Datasets as Topic , Dendritic Cells/immunology , Disease Models, Animal , Enzyme Inhibitors/pharmacology , Extracellular Vesicles/metabolism , Female , Humans , Kaplan-Meier Estimate , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Mice , Middle Aged , Ovarian Neoplasms/blood , Ovarian Neoplasms/mortality , Ovarian Neoplasms/pathology
3.
Leukemia ; 33(10): 2416-2428, 2019 10.
Article in English | MEDLINE | ID: mdl-30872780

ABSTRACT

The BCL-2 inhibitor venetoclax has only limited activity in DLBCL despite frequent BCL-2 overexpression. Since constitutive activation of the B cell receptor (BCR) pathway has been reported in both ABC and GCB DLBCL, we investigated whether targeting SYK or BTK will increase sensitivity of DLBCL cells to venetoclax. We report that pharmacological inhibition of SYK or BTK synergistically enhances venetoclax sensitivity in BCL-2-positive DLBCL cell lines with an activated BCR pathway in vitro and in a xenograft model in vivo, despite the only modest direct cytotoxic effect. We further show that these sensitizing effects are associated with inhibition of the downstream PI3K/AKT pathway and changes in the expression of MCL-1, BIM, and HRK. In addition, we show that BCR-dependent GCB DLBCL cells are characterized by deficiency of the phosphatase SHP1, a key negative regulator of the BCR pathway. Re-expression of SHP1 in GCB DBLCL cells reduces SYK, BLNK, and GSK3 phosphorylation and induces corresponding changes in MCL1, BIM, and HRK expression. Together, these findings suggest that SHP1 deficiency is responsible for the constitutive activation of the BCR pathway in GCB DLBCL and identify SHP1 and BCL-2 as potential predictive markers for response to treatment with a venetoclax/BCR inhibitor combination.


Subject(s)
Agammaglobulinaemia Tyrosine Kinase/metabolism , Antineoplastic Agents/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Lymphoma, Large B-Cell, Diffuse/drug therapy , Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Sulfonamides/pharmacology , Syk Kinase/metabolism , Animals , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Humans , Lymphoma, Large B-Cell, Diffuse/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction/drug effects , Xenograft Model Antitumor Assays/methods
4.
Oncoimmunology ; 7(5): e1423183, 2018.
Article in English | MEDLINE | ID: mdl-29721381

ABSTRACT

Diminished overall survival rate of non-Hodgkin lymphoma (NHL) patients treated with a combination regimen of rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) has been recently linked to recurrent somatic mutations activating FOXO1. Despite of the clinical relevance of this finding, the molecular mechanism driving resistance to R-CHOP therapy remains largely unknown. Herein, we investigated the potential role of FOXO1 in the therapeutic efficacy of rituximab, the only targeted therapy included in the R-CHOP regimen. We found CD20 transcription is negatively regulated by FOXO1 in NHL cell lines and in human lymphoma specimens carrying activating mutations of FOXO1. Furthermore, both the expression of exogenous mutants of FOXO1 and the inhibition of AKT led to FOXO1 activation in lymphoma cells, increased binding to MS4A1 promoter and diminished CD20 expression levels. In contrast, a disruption of FOXO1 with CRISPR/Cas9 genome-editing (sgFOXO1) resulted in CD20 upregulation, improved the cytotoxicity induced by rituximab and the survival of mice with sgFOXO1 tumors. Accordingly, pharmacological inhibition of FOXO1 activity in primary samples upregulated surface CD20 levels. Importantly, FOXO1 was required for the downregulation of CD20 levels by the clinically tested inhibitors of BTK, SYK, PI3K and AKT. Taken together, these results indicate for the first time that the AKT-unresponsive mutants of FOXO1 are important determinant of cell response to rituximab-induced cytotoxicity, and suggest that the genetic status of FOXO1 together with its transcriptional activity need further attention while designing anti-CD20 antibodies based regimens for the therapy of pre-selected lymphomas.

5.
Blood ; 130(14): 1628-1638, 2017 10 05.
Article in English | MEDLINE | ID: mdl-28830887

ABSTRACT

Downregulation of CD20, a molecular target for monoclonal antibodies (mAbs), is a clinical problem leading to decreased efficacy of anti-CD20-based therapeutic regimens. The epigenetic modulation of CD20 coding gene (MS4A1) has been proposed as a mechanism for the reduced therapeutic efficacy of anti-CD20 antibodies and confirmed with nonselective histone deacetylase inhibitors (HDACis). Because the use of pan-HDACis is associated with substantial adverse effects, the identification of particular HDAC isoforms involved in CD20 regulation seems to be of paramount importance. In this study, we demonstrate for the first time the role of HDAC6 in the regulation of CD20 levels. We show that inhibition of HDAC6 activity significantly increases CD20 levels in established B-cell tumor cell lines and primary malignant cells. Using pharmacologic and genetic approaches, we confirm that HDAC6 inhibition augments in vitro efficacy of anti-CD20 mAbs and improves survival of mice treated with rituximab. Mechanistically, we demonstrate that HDAC6 influences synthesis of CD20 protein independently of the regulation of MS4A1 transcription. We further demonstrate that translation of CD20 mRNA is significantly enhanced after HDAC6 inhibition, as shown by the increase of CD20 mRNA within the polysomal fraction, indicating a new role of HDAC6 in the posttranscriptional mechanism of CD20 regulation. Collectively, our findings suggest HDAC6 inhibition is a rational therapeutic strategy to be implemented in combination therapies with anti-CD20 monoclonal antibodies and open up novel avenues for the clinical use of HDAC6 inhibitors.


Subject(s)
Antigens, CD20/genetics , Antineoplastic Agents/pharmacology , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Lymphoma, Non-Hodgkin/drug therapy , Rituximab/pharmacology , Animals , Antigens, CD20/immunology , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , B-Lymphocytes/pathology , Cell Line, Tumor , Female , Gene Expression Regulation, Neoplastic/drug effects , Histone Deacetylase 6 , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Lymphoma, Non-Hodgkin/genetics , Lymphoma, Non-Hodgkin/immunology , Lymphoma, Non-Hodgkin/pathology , Mice, Inbred BALB C , Mice, SCID , RNA, Messenger/genetics , Tumor Cells, Cultured , Up-Regulation/drug effects
6.
Cancer Res ; 74(5): 1371-8, 2014 Mar 01.
Article in English | MEDLINE | ID: mdl-24398474

ABSTRACT

How necrotic areas develop in tumors is incompletely understood but can impact progression. Recent findings suggest that the formation of vascular microthrombi contributes to tumor necrosis, prompting investigation of coagulation cascades. Here, we report that loss of tumor suppressor P14ARF can contribute to activating the clotting cascade in glioblastoma. P14ARF transcriptionally upregulated TFPI2, a Kunitz-type serine protease in the tissue factor pathway that inhibits the initiation of thrombosis reactions. P14ARF activation in tumor cells delayed their ability to activate plasma clotting. Mechanistically, P14ARF activated the TFPI2 promoter in a p53-independent manner that relied upon c-JUN, SP1, and JNK activity. Taken together, our results identify the critical signaling pathways activated by P14ARF to prevent vascular microthrombosis triggered by glioma cells. Stimulation of this pathway might be used as a therapeutic strategy to reduce aggressive phenotypes associated with necrotic tumors, including glioblastoma.


Subject(s)
Glioblastoma/genetics , Signal Transduction/genetics , Thromboplastin/genetics , Thrombosis/genetics , Tumor Suppressor Protein p14ARF/genetics , Genes, Tumor Suppressor , Humans , Promoter Regions, Genetic/genetics , Transcriptional Activation/genetics , Tumor Cells, Cultured , Up-Regulation/genetics
7.
J Clin Invest ; 122(4): 1283-95, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22378045

ABSTRACT

Malignant gliomas are the most common and the most lethal primary brain tumors in adults. Among malignant gliomas, 60%-80% show loss of P14ARF tumor suppressor activity due to somatic alterations of the INK4A/ARF genetic locus. The tumor suppressor activity of P14ARF is in part a result of its ability to prevent the degradation of P53 by binding to and sequestering HDM2. However, the subsequent finding of P14ARF loss in conjunction with TP53 gene loss in some tumors suggests the protein may have other P53-independent tumor suppressor functions. Here, we report what we believe to be a novel tumor suppressor function for P14ARF as an inhibitor of tumor-induced angiogenesis. We found that P14ARF mediates antiangiogenic effects by upregulating expression of tissue inhibitor of metalloproteinase-3 (TIMP3) in a P53-independent fashion. Mechanistically, this regulation occurred at the gene transcription level and was controlled by HDM2-SP1 interplay, where P14ARF relieved a dominant negative interaction of HDM2 with SP1. P14ARF-induced expression of TIMP3 inhibited endothelial cell migration and vessel formation in response to angiogenic stimuli produced by cancer cells. The discovery of this angiogenesis regulatory pathway may provide new insights into P53-independent P14ARF tumor-suppressive mechanisms that have implications for the development of novel therapies directed at tumors and other diseases characterized by vascular pathology.


Subject(s)
Brain Neoplasms/blood supply , Gene Expression Regulation, Neoplastic , Glioblastoma/blood supply , Neoplasm Proteins/physiology , Neovascularization, Pathologic/physiopathology , Tissue Inhibitor of Metalloproteinase-3/biosynthesis , Tumor Suppressor Protein p14ARF/physiology , Animals , Brain Neoplasms/metabolism , Cell Line, Transformed , Chemotaxis/drug effects , Corneal Neovascularization/physiopathology , Culture Media, Conditioned/pharmacology , Endothelial Cells/pathology , Fibroblasts/drug effects , Genes, p53 , Glioblastoma/metabolism , Humans , Mice , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Neovascularization, Pathologic/genetics , Proto-Oncogene Proteins c-mdm2/physiology , RNA Interference , RNA, Small Interfering/pharmacology , Recombinant Fusion Proteins/physiology , Sp1 Transcription Factor/physiology , Tissue Inhibitor of Metalloproteinase-3/antagonists & inhibitors , Tissue Inhibitor of Metalloproteinase-3/genetics , Tissue Inhibitor of Metalloproteinase-3/physiology , Transcription, Genetic , Tumor Cells, Cultured/metabolism , Up-Regulation
9.
PLoS One ; 2(6): e576, 2007 Jun 27.
Article in English | MEDLINE | ID: mdl-17593975

ABSTRACT

The structural complexity of chromosome 1p centromeric region has been an obstacle for fine mapping of tumor suppressor genes in this area. Loss of heterozygosity (LOH) on chromosome 1p is associated with the longer survival of oligodendroglioma (OD) patients. To test the clinical relevance of 1p loss in glioblastomas (GBM) patients and identifiy the underlying tumor suppressor locus, we constructed a somatic deletion map on chromosome 1p in 26 OG and 118 GBM. Deletion hotspots at 4 microsatellite markers located at 1p36.3, 1p36.1, 1p22 and 1p11 defined 10 distinct haplotypes that were related to patient survival. We found that loss of 1p centromeric marker D1S2696 within NOTCH2 intron 12 was associated with favorable prognosis in OD (P = 0.0007) as well as in GBM (P = 0.0175), while 19q loss, concomitant with 1p LOH in OD, had no influence on GBM survival (P = 0.918). Assessment of the intra-chromosomal ratio between NOTCH2 and its 1q21 pericentric duplication N2N (N2/N2N-test) allowed delineation of a consistent centromeric breakpoint in OD that also contained a minimally lost area in GBM. OD and GBM showed distinct deletion patterns that converged to the NOTCH2 gene in both glioma subtypes. Moreover, the N2/N2N-test disclosed homozygous deletions of NOTCH2 in primary OD. The N2/N2N test distinguished OD from GBM with a specificity of 100% and a sensitivity of 97%. Combined assessment of NOTCH2 genetic markers D1S2696 and N2/N2N predicted 24-month survival with an accuracy (0.925) that is equivalent to histological classification combined with the D1S2696 status (0.954) and higher than current genetic evaluation by 1p/19q LOH (0.762). Our data propose NOTCH2 as a powerful new molecular test to detect prognostically favorable gliomas.


Subject(s)
Brain Neoplasms/genetics , Chromosome Deletion , Chromosomes, Human, Pair 1/genetics , Glioma/genetics , Receptor, Notch2/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Brain Neoplasms/mortality , Brain Neoplasms/pathology , Child , Child, Preschool , Female , Genes, Tumor Suppressor , Glioma/mortality , Glioma/pathology , Humans , Infant , Male , Microsatellite Repeats , Middle Aged , Survival Rate , Young Adult
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