ABSTRACT
The ternary complex (eIF2·GTP·Met-tRNAiMet) and the eIF4F complex assembly are two major regulatory steps in the eukaryotic translation initiation. Inhibition of the ternary complex assembly is therefore a promising target for the development of novel anti-cancer therapeutics. Building on the finding that clotrimazole (CLT), a molecular probe that depletes intracellular Ca2+ stores and subsequently induce eIF2α phosphorylation, inhibit translation initiation, and reduce preferentially the expression of oncoproteins over "housekeeping" ones,1-3 we undertook structure activity relationship (SAR) studies that identified 3,3-diarylindoline-2-one #1181 as an interesting scaffold. Compound #1181 also induce phosphorylation of eIF2α thereby reducing the availability of the ternary complex, which leads to inhibition of translation initiation.4 Our subsequent efforts focused on understanding SAR iterative lead optimization to enhance potency and improve bioavailability. Herein, we report a complementing study focusing on heavily substituted symmetric and asymmetric 3,3-(o,m-disubstituted)diarylindoline-2-ones. These compounds were evaluated by the dual luciferase reporter ternary complex assay that recapitualates phosphorylation of eIF2α in a quantitative manner. We also evaluated all compounds by sulforhodamine B assay, which measures the overall effect of compounds on cell proliferations and/or viability.
Subject(s)
Biphenyl Compounds , Eukaryotic Initiation Factor-2 , Eukaryotic Initiation Factor-2/genetics , Eukaryotic Initiation Factor-2/metabolism , Phosphorylation , Protein BiosynthesisABSTRACT
CD38 is a transmembrane glycoprotein that is widely expressed in a variety of human tissues and cells, especially those in the immune system. CD38 protein was previously considered as a cell activation marker, and today monoclonal antibodies targeting CD38 have witnessed great achievements in multiple myeloma and promoted researchers to conduct research on other tumors. In this review, we provide a wide-ranging review of the biology and function of the human molecule outside the field of myeloma. We focus mainly on current research findings to summarize and update the findings gathered from diverse areas of study. Based on these findings, we attempt to extend the role of CD38 in the context of therapy of solid tumors and expand the role of the molecule from a simple marker to an immunomodulator.
Subject(s)
ADP-ribosyl Cyclase 1/physiology , Immunologic Factors/physiology , Neoplasms/immunology , ADP-ribosyl Cyclase 1/analysis , ADP-ribosyl Cyclase 1/antagonists & inhibitors , Antibodies, Monoclonal/therapeutic use , Humans , ImmunotherapyABSTRACT
OBJECTIVE: High grade serous ovarian cancer (HGSC) remains one of the most lethal malignancies in females. We previously reported that γ-glutamyl cyclotransferase (GGCT) was significantly upregulated in serous ovarian cancer. The current study was aimed to explore the function and underlying mechanism of GGCT in HGSC. METHODS: GGCT expression was assessed by immunohistochemistry in 128 HGSC patients. Stable cell lines with GGCT gene overexpression or knockdown were established to investigate the function of GGCT in HGSC in vitro and in vivo. RESULTS: GGCT is highly upregulated in HGSC tissues and associated with FIGO stage, lymph node metastasis and ascitic fluid volume. High expression of GGCT is associated with poor survival in HGSC patients. The Harrell's c-indexes of the prognostic models for overall survival and progression-free survival prediction were 0.758 and 0.726, respectively. GGCT knockdown suppresses proliferation, clone formation, migration, and invasion of tumor cells in vitro while forced GGCT overexpression presents opposite results. Furthermore, GGCT silencing inhibits tumor growth and spread in vivo. Epithelial-mesenchymal transition (EMT) and PI3K/AKT/mTOR signaling pathway are suppressed in GGCT silenced cells and enhanced in GGCT overexpressed cells. Inactivation of PI3K/AKT/mTOR signaling pathway in GGCT overexpressed cells induces EMT inhibition. CONCLUSIONS: Our data reveals an important role of GGCT in regulating EMT and progression of HGSC, providing a valuable prognostic marker and potential target for treatment of HGSC patients.
Subject(s)
Cystadenocarcinoma, Serous/enzymology , Ovarian Neoplasms/enzymology , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , TOR Serine-Threonine Kinases/metabolism , gamma-Glutamylcyclotransferase/biosynthesis , Animals , Cell Proliferation/physiology , Cystadenocarcinoma, Serous/pathology , Disease Progression , Epithelial-Mesenchymal Transition , Female , Heterografts , Humans , Immunohistochemistry , Mice , Mice, Inbred BALB C , Neoplasm Grading , Neoplasm Invasiveness , Neoplasm Metastasis , Ovarian Neoplasms/pathology , Signal Transduction , Up-Regulation , gamma-Glutamylcyclotransferase/metabolismABSTRACT
4EGI-1, the prototypic inhibitor of eIF4E/eIF4G interaction, was identified in a high-throughput screening of small-molecule libraries with the aid of a fluorescence polarization assay that measures inhibition of binding of an eIF4G-derived peptide to recombinant eIF4E. As such, the molecular probe 4EGI-1 has potential for the study of molecular mechanisms involved in human disorders characterized by loss of physiological restraints on translation initiation. A hit-to-lead optimization campaign was carried out to overcome the configurational instability in 4EGI-1, which stems from the E-to-Z isomerization of the hydrazone function. We identified compound 1 a, in which the labile hydrazone was incorporated into a rigid indazole scaffold, as a promising rigidified 4EGI-1 mimetic lead. In a structure-activity relationship study directed towards probing the structural latitude of this new chemotype as an inhibitor of eIF4E/eIF4G interaction and translation initiation we identified 1 d, an indazole-based 4EGI-1 mimetic, as a new and improved lead inhibitor of eIF4E/eIF4G interaction and a promising molecular probe candidate for elucidation of the role of cap-dependent translation initiation in a host of pathophysiological states.
Subject(s)
Eukaryotic Initiation Factor-4E/metabolism , Eukaryotic Initiation Factor-4G/metabolism , Hydrazones/metabolism , Indazoles/chemistry , Thiazoles/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Eukaryotic Initiation Factor-4E/antagonists & inhibitors , Eukaryotic Initiation Factor-4G/antagonists & inhibitors , Humans , Hydrazones/chemistry , Hydrazones/pharmacology , Indazoles/chemical synthesis , Indazoles/pharmacology , Protein Binding , Protein Interaction Domains and Motifs/drug effects , Stereoisomerism , Structure-Activity Relationship , Thiazoles/chemistry , Thiazoles/pharmacologyABSTRACT
Translation initiation plays a critical role in cellular homeostasis, proliferation, differentiation and malignant transformation. Consistently, increasing the abundance of the eIF2-GTP-tRNA(i)(Met) translation initiation complex transforms normal cells and contributes to cancer initiation and the severity of some anemias. The chemical modifiers of the eIF2-GTP-tRNA(i)(Met) ternary complex are therefore invaluable tools for studying its role in the pathobiology of human disorders and for determining whether this complex can be pharmacologically targeted for therapeutic purposes. Using a cell-based assay, we identified N,N'-diarylureas as unique inhibitors of ternary complex accumulation. Direct functional-genetic and biochemical evidence demonstrated that the N,N'-diarylureas activate heme-regulated inhibitor kinase, thereby phosphorylating eIF2α and reducing the abundance of the ternary complex. Using tumor cell proliferation in vitro and tumor growth in vivo as paradigms, we demonstrate that N,N'-diarylureas are potent and specific tools for studying the role of eIF2-GTP-tRNA(i)(Met) ternary complex in the pathobiology of human disorders.
Subject(s)
Antineoplastic Agents/chemistry , Peptide Chain Initiation, Translational/drug effects , Urea/analogs & derivatives , eIF-2 Kinase/antagonists & inhibitors , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Male , Mice , Mice, Nude , Xenograft Model Antitumor Assays , eIF-2 Kinase/chemistry , eIF-2 Kinase/geneticsABSTRACT
Introduction: Despite significant clinical advancement with the use of immune checkpoint blockade (ICB) in non-small cell lung cancer (NSCLC) there are still a major subset of patients that develop adaptive/acquired resistance. Understanding resistance mechanisms to ICB is critical to developing new therapeutic strategies and improving patient survival. The dynamic nature of the tumor microenvironment and the mutational load driving tumor immunogenicity limit the efficacy to ICB. Recent studies indicate that myeloid cells are drivers of ICB resistance. In this study we sought to understand which immune cells were contributing to resistance and if we could modify them in a way to improve response to ICB therapy. Results: Our results show that combination anti-PD-1/CTLA-4 produces an initial antitumor effect with evidence of an activated immune response. Upon extended treatment with anti-PD-1/CTLA-4 acquired resistance developed with an increase of the immunosuppressive populations, including T-regulatory cells, neutrophils and monocytes. Addition of anti-Ly6C blocking antibody to anti-PD-1/CTLA-4 was capable of completely reversing treatment resistance and restoring CD8 T cell activity in multiple KP lung cancer models and in the autochthonous lung cancer KrasLSL-G12D/p53fl/fl model. We found that there were higher classical Ly6C+ monocytes in anti-PD-1/CTLA-4 combination resistant tumors. B7 blockade illustrated the importance of dendritic cells for treatment efficacy of anti-Ly6C/PD-1/CTLA-4. We further determined that classical Ly6C+ monocytes in anti-PD-1/CTLA-4 resistant tumors are trafficked into the tumor via IFN-γ and the CCL2-CCR2 axis. Mechanistically we found that classical monocytes from ICB resistant tumors were unable to differentiate into antigen presenting cells and instead differentiated into immunosuppressive M2 macrophages or myeloid-derived suppressor cells (MDSC). Classical Ly6C+ monocytes from ICB resistant tumors had a decrease in both Flt3 and PU.1 expression that prevented differentiation into dendritic cells/macrophages. Conclusions: Therapeutically we found that addition of anti-Ly6C to the combination of anti-PD-1/CTLA-4 was capable of complete tumor eradication. Classical Ly6C+ monocytes differentiate into immunosuppressive cells, while blockade of classical monocytes drives dendritic cell differentiation/maturation to reinvigorate the anti-tumor T cell response. These findings support that immunotherapy resistance is associated with infiltrating monocytes and that controlling the differentiation process of monocytes can enhance the therapeutic potential of ICB.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Humans , Monocytes , CTLA-4 Antigen , Carcinoma, Non-Small-Cell Lung/therapy , Lung Neoplasms/therapy , Immunotherapy/methods , Tumor MicroenvironmentABSTRACT
Symmetrical N,N'-diarylureas: 1,3-bis(3,4-dichlorophenyl)-, 1,3-bis[4-chloro-3-(trifluoromethyl)phenyl]- and 1,3-bis[3,5-bis(trifluoromethyl)phenyl]urea, were identified as potent activators of the eIF2α kinase heme regulated inhibitor. They reduce the abundance of the eIF2·GTP·tRNA(i)(Met) ternary complex and inhibit cancer cell proliferation. An optimization process was undertaken to improve their solubility while preserving their biological activity. Non-symmetrical hybrid ureas were generated by combining one of the hydrophobic phenyl moieties present in the symmetrical ureas with the polar 3-hydroxy-tolyl moiety. O-alkylation of the later added potentially solubilizing charge bearing groups. The new non-symmetrical N,N'-diarylureas were characterized by ternary complex reporter gene and cell proliferation assays, demonstrating good bioactivities. A representative sample of these compounds potently induced phosphorylation of eIF2α and expression of CHOP at the protein and mRNA levels. These inhibitors of translation initiation may become leads for the development of potent, non-toxic, and target specific anti-cancer agents.
Subject(s)
Antineoplastic Agents/pharmacology , Chemistry, Pharmaceutical/methods , Animals , Antineoplastic Agents/chemical synthesis , Cell Line, Tumor , Cell Proliferation , Drug Design , Eukaryotic Initiation Factor-2/chemistry , Genes, Reporter , Humans , In Vitro Techniques , Inhibitory Concentration 50 , Mice , Models, Chemical , Phosphorylation , RNA, Messenger/metabolism , RNA, Transfer, Met/chemistry , Structure-Activity Relationship , Transcription Factor CHOP/chemistry , Transfection , Urea/chemistryABSTRACT
CXCL14 is a relatively new chemokine with unidentified receptor and undefined function. Recently, we found that CXCL14 is upregulated in arthritic joints in a mouse model of autoimmune arthritis, collagen-induced arthritis. To examine the role of CXCL14 in the development and pathogenesis of autoimmune arthritis, we have generated transgenic (Tg) mice that overexpress CXCL14 under control of phosphoglycerate kinase promoter. The results showed that CXCL14-Tg mice developed more severe arthritis compared with wild-type controls. The draining lymph nodes of CXCL14-Tg mice were significantly enlarged and contained an increased number of activated T cells, particularly the CD44(+)CD62L(low) effector memory cells. In addition, T cells from CXCL14-Tg mice exhibited an enhanced proliferative response against collagen II and produced higher levels of IFN-gamma but not IL-4 or IL-17. CXCL14-Tg mice also had elevated levels of IgG2a autoantibodies. These findings indicated that CXCL14 plays an important role in the autoimmune arthritis, which may have an implication in understanding the pathogenic mechanisms of rheumatoid arthritis in humans and, ultimately, therapeutic interference.
Subject(s)
Arthritis, Experimental/immunology , Arthritis, Experimental/pathology , Chemokines, CXC/biosynthesis , Chemokines, CXC/genetics , Animals , Arthritis, Experimental/genetics , Autoantibodies/biosynthesis , Autoantibodies/physiology , Chemokines, CXC/physiology , Chickens , Collagen Type II/toxicity , Cytokines/biosynthesis , Cytokines/physiology , Immunoglobulin G/biosynthesis , Immunoglobulin M/biosynthesis , Inflammation Mediators/physiology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Promoter Regions, Genetic/immunology , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/physiology , Severity of Illness Index , Th1 Cells/immunology , Th1 Cells/metabolism , Th1 Cells/pathology , Up-Regulation/genetics , Up-Regulation/immunologyABSTRACT
Understanding resistance mechanisms to targeted therapies and immune checkpoint blockade in mutant KRAS lung cancers is critical to developing novel combination therapies and improving patient survival. Here, we show that MEK inhibition enhanced PD-L1 expression while PD-L1 blockade upregulated MAPK signaling in mutant KRAS lung tumors. Combined MEK inhibition with anti-PD-L1 synergistically reduced lung tumor growth and metastasis, but tumors eventually developed resistance to sustained combinatorial therapy. Multi-platform profiling revealed that resistant lung tumors have increased infiltration of Th17 cells, which secrete IL-17 and IL-22 cytokines to promote lung cancer cell invasiveness and MEK inhibitor resistance. Antibody depletion of IL-17A in combination with MEK inhibition and PD-L1 blockade markedly reduced therapy-resistance in vivo. Clinically, increased expression of Th17-associated genes in patients treated with PD-1 blockade predicted poorer overall survival and response in melanoma and predicated poorer response to anti-PD1 in NSCLC patients. Here we show a triple combinatorial therapeutic strategy to overcome resistance to combined MEK inhibitor and PD-L1 blockade.
Subject(s)
Antineoplastic Agents, Immunological/pharmacology , B7-H1 Antigen/metabolism , Carcinoma, Non-Small-Cell Lung/drug therapy , Lung Neoplasms/drug therapy , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Proto-Oncogene Proteins p21(ras)/genetics , Th17 Cells/metabolism , Tumor Suppressor Protein p53/genetics , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , B7-H1 Antigen/immunology , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/immunology , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/mortality , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Drug Resistance, Neoplasm/immunology , Drug Synergism , Female , Humans , Immune Checkpoint Inhibitors/immunology , Immunohistochemistry , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/genetics , Male , Mice , Mice, Knockout , Mitogen-Activated Protein Kinase Kinases/metabolism , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/immunology , Neoplasm Metastasis , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins p21(ras)/metabolism , Th17 Cells/immunology , Tumor Suppressor Protein p53/metabolismABSTRACT
We have previously demonstrated that PD-1 blockade decreased the incidence of high-grade dysplasia in a carcinogen-induced murine model of oral squamous cell carcinoma (OSCC). It remains unknown, however, whether there are additional factors involved in escape from immune surveillance that could serve as additional targets for immunoprevention. We performed this study to further characterize the immune landscape of oral premalignant lesions (OPL) and determine the impact of targeting of the PD-1, CTLA-4, CD40, or OX40 pathways on the development of OPLs and oral carcinomas in the 4-nitroquinoline 1-oxide model. The immune pathways were targeted using mAbs or, in the case of the PD-1/PD-L1 pathway, using PD-L1-knockout (PD-L1ko) mice. After intervention, tongues and cervical lymph nodes were harvested and analyzed for malignant progression and modulation of the immune milieu, respectively. Targeting of CD40 with an agonist mAb was the most effective treatment to reduce transition of OPLs to OSCC; PD-1 alone or in combination with CTLA-4 inhibition, or PD-L1ko, also reduced progression of OPLs to OSCC, albeit to a lesser extent. Distinct patterns of immune system modulation were observed for the CD40 agonists compared with blockade of the PD-1/PD-L1 axis with or without CTLA-4 blockade; CD40 agonist generated a lasting expansion of experienced/memory cytotoxic T lymphocytes and M1 macrophages, whereas PD-1/CTLA-4 blockade resulted in a pronounced depletion of regulatory T cells among other changes. These data suggest that distinct approaches may be used for targeting different steps in the development of OSCC, and that CD40 agonists merit investigation as potential immunoprevention agents in this setting. PREVENTION RELEVANCE: PD-1/PD-L1 pathway blockade, as well as activation of the CD40 pathway, were able to prevent OPL progression into invasive OSCC in a murine model. A distinct pattern of immune modulation was observed when either the CD40 or the PD-1/PD-L1 pathways were targeted.
Subject(s)
Antibodies, Monoclonal/pharmacology , B7-H1 Antigen/antagonists & inhibitors , CD40 Antigens/antagonists & inhibitors , Carcinoma, Squamous Cell/drug therapy , Immune Checkpoint Inhibitors/pharmacology , Mouth Neoplasms/drug therapy , Precancerous Conditions/drug therapy , 4-Nitroquinoline-1-oxide/toxicity , Animals , Carcinogens/toxicity , Carcinoma, Squamous Cell/chemically induced , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/pathology , Female , Immunotherapy , Mice , Mice, Inbred C57BL , Mouth Neoplasms/chemically induced , Mouth Neoplasms/metabolism , Mouth Neoplasms/pathology , Precancerous Conditions/chemically induced , Precancerous Conditions/metabolism , Precancerous Conditions/pathologyABSTRACT
Tumor extracellular matrix has been associated with drug resistance and immune suppression. Here, proteomic and RNA profiling reveal increased collagen levels in lung tumors resistant to PD-1/PD-L1 blockade. Additionally, elevated collagen correlates with decreased total CD8+ T cells and increased exhausted CD8+ T cell subpopulations in murine and human lung tumors. Collagen-induced T cell exhaustion occurs through the receptor LAIR1, which is upregulated following CD18 interaction with collagen, and induces T cell exhaustion through SHP-1. Reduction in tumor collagen deposition through LOXL2 suppression increases T cell infiltration, diminishes exhausted T cells, and abrogates resistance to anti-PD-L1. Abrogating LAIR1 immunosuppression through LAIR2 overexpression or SHP-1 inhibition sensitizes resistant lung tumors to anti-PD-1. Clinically, increased collagen, LAIR1, and TIM-3 expression in melanoma patients treated with PD-1 blockade predict poorer survival and response. Our study identifies collagen and LAIR1 as potential markers for immunotherapy resistance and validates multiple promising therapeutic combinations.
Subject(s)
Adenocarcinoma of Lung/drug therapy , Antineoplastic Agents, Immunological/pharmacology , CD8-Positive T-Lymphocytes/immunology , Collagen/metabolism , Drug Resistance, Neoplasm/immunology , Lung Neoplasms/drug therapy , Receptors, Immunologic/metabolism , Adenocarcinoma of Lung/genetics , Adenocarcinoma of Lung/immunology , Adenocarcinoma of Lung/pathology , Amino Acid Oxidoreductases/genetics , Amino Acid Oxidoreductases/metabolism , Animals , Antineoplastic Agents, Immunological/therapeutic use , B7-H1 Antigen/antagonists & inhibitors , B7-H1 Antigen/immunology , Biomarkers, Tumor/metabolism , Carcinoma, Lewis Lung/drug therapy , Carcinoma, Lewis Lung/immunology , Carcinoma, Lewis Lung/pathology , Cell Line, Tumor , Datasets as Topic , Disease Models, Animal , Extracellular Matrix/drug effects , Extracellular Matrix/immunology , Extracellular Matrix/pathology , Female , Gene Knockdown Techniques , HEK293 Cells , Hepatitis A Virus Cellular Receptor 2/metabolism , Humans , Lung/immunology , Lung/pathology , Lung Neoplasms/genetics , Lung Neoplasms/immunology , Lung Neoplasms/pathology , Male , Mice , Mice, Transgenic , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Programmed Cell Death 1 Receptor/immunology , Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , RNA-Seq , Receptors, Immunologic/geneticsABSTRACT
Despite recent advances in the use of immunotherapy, only a minority of patients with small cell lung cancer (SCLC) respond to immune checkpoint blockade (ICB). Here, we show that targeting the DNA damage response (DDR) proteins PARP and checkpoint kinase 1 (CHK1) significantly increased protein and surface expression of PD-L1. PARP or CHK1 inhibition remarkably potentiated the antitumor effect of PD-L1 blockade and augmented cytotoxic T-cell infiltration in multiple immunocompetent SCLC in vivo models. CD8+ T-cell depletion reversed the antitumor effect, demonstrating the role of CD8+ T cells in combined DDR-PD-L1 blockade in SCLC. We further demonstrate that DDR inhibition activated the STING/TBK1/IRF3 innate immune pathway, leading to increased levels of chemokines such as CXCL10 and CCL5 that induced activation and function of cytotoxic T lymphocytes. Knockdown of cGAS and STING successfully reversed the antitumor effect of combined inhibition of DDR and PD-L1. Our results define previously unrecognized innate immune pathway-mediated immunomodulatory functions of DDR proteins and provide a rationale for combining PARP/CHK1 inhibitors and immunotherapies in SCLC. SIGNIFICANCE: Our results define previously unrecognized immunomodulatory functions of DDR inhibitors and suggest that adding PARP or CHK1 inhibitors to ICB may enhance treatment efficacy in patients with SCLC. Furthermore, our study supports a role of innate immune STING pathway in DDR-mediated antitumor immunity in SCLC.See related commentary by Hiatt and MacPherson, p. 584.This article is highlighted in the In This Issue feature, p. 565.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , DNA Damage , Lung Neoplasms/drug therapy , Lung Neoplasms/immunology , Membrane Proteins/immunology , Small Cell Lung Carcinoma/drug therapy , Small Cell Lung Carcinoma/immunology , Animals , Apoptosis/drug effects , B7-H1 Antigen/antagonists & inhibitors , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/metabolism , Cell Proliferation/drug effects , Female , Humans , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/metabolism , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Lymphocyte Activation/drug effects , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Nude , Phthalazines/pharmacology , Piperazines/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Pyrazines/pharmacology , Pyrazoles/pharmacology , Random Allocation , Small Cell Lung Carcinoma/genetics , Small Cell Lung Carcinoma/pathology , T-Lymphocytes, Cytotoxic/drug effects , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolism , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Although treatment with immune checkpoint inhibitors provides promising benefit for patients with cancer, optimal use is encumbered by high resistance rates and requires a thorough understanding of resistance mechanisms. We observed that tumors treated with PD-1/PD-L1 blocking antibodies develop resistance through the upregulation of CD38, which is induced by all-trans retinoic acid and IFNß in the tumor microenvironment. In vitro and in vivo studies demonstrate that CD38 inhibits CD8+ T-cell function via adenosine receptor signaling and that CD38 or adenosine receptor blockade are effective strategies to overcome the resistance. Large data sets of human tumors reveal expression of CD38 in a subset of tumors with high levels of basal or treatment-induced T-cell infiltration, where immune checkpoint therapies are thought to be most effective. These findings provide a novel mechanism of acquired resistance to immune checkpoint therapy and an opportunity to expand their efficacy in cancer treatment.Significance: CD38 is a major mechanism of acquired resistance to PD-1/PD-L1 blockade, causing CD8+ T-cell suppression. Coinhibition of CD38 and PD-L1 improves antitumor immune response. Biomarker assessment in patient cohorts suggests that a combination strategy is applicable to a large percentage of patients in whom PD-1/PD-L1 blockade is currently indicated. Cancer Discov; 8(9); 1156-75. ©2018 AACR.See related commentary by Mittal et al., p. 1066This article is highlighted in the In This Issue feature, p. 1047.
Subject(s)
ADP-ribosyl Cyclase 1/metabolism , Antineoplastic Agents, Immunological/administration & dosage , Carcinoma, Non-Small-Cell Lung/drug therapy , Drug Resistance, Neoplasm/drug effects , Lung Neoplasms/drug therapy , Melanoma/drug therapy , Membrane Glycoproteins/metabolism , ADP-ribosyl Cyclase 1/antagonists & inhibitors , Animals , Antineoplastic Agents, Immunological/pharmacology , B7-H1 Antigen/antagonists & inhibitors , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , Carcinoma, Non-Small-Cell Lung/immunology , Cell Line, Tumor , Drug Synergism , Gene Expression Regulation, Neoplastic/drug effects , Humans , Interferon-gamma/metabolism , Lung Neoplasms/immunology , Melanoma/genetics , Melanoma/immunology , Membrane Glycoproteins/antagonists & inhibitors , Mice , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Receptors, Purinergic P1/metabolism , Signal Transduction/drug effects , Tretinoin/metabolism , Tumor Microenvironment/drug effects , Up-Regulation/drug effects , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: Promising results in the treatment of non-small cell lung cancer (NSCLC) have been seen with agents targeting immune checkpoints, such as programmed cell death 1 (PD-1) or programmed death ligand-1 (PD-L1). However, only a select group of patients respond to these interventions. The identification of biomarkers that predict clinical benefit to immune checkpoint blockade is critical to successful clinical translation of these agents. METHODS: We conducted an integrated analysis of three independent large datasets, including The Cancer Genome Atlas of lung adenocarcinoma and two datasets from MD Anderson Cancer Center (Houston, TX), Profiling of Resistance Patterns and Oncogenic Signaling Pathways in Evaluation of Cancers of the Thorax (named PROSPECT) and Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination (named BATTLE-1). Comprehensive analysis of mRNA gene expression, reverse-phase protein array, IHC, and correlation with clinical data were performed. RESULTS: Epithelial-mesenchymal transition (EMT) is highly associated with an inflammatory tumor microenvironment in lung adenocarcinoma, independent of tumor mutational burden. We found immune activation coexistent with elevation of multiple targetable immune checkpoint molecules, including PD-L1, PD-L2, PD-1, TIM-3, B7-H3, BTLA, and CTLA-4, along with increases in tumor infiltration by CD4(+)Foxp3(+) regulatory T cells in lung adenocarcinomas that displayed an EMT phenotype. Furthermore, we identify B7-H3 as a prognostic marker for NSCLC. CONCLUSIONS: The strong association between EMT status and an inflammatory tumor microenvironment with elevation of multiple targetable immune checkpoint molecules warrants further investigation of using EMT as a predictive biomarker for immune checkpoint blockade agents and other immunotherapies in NSCLC and possibly a broad range of other cancers. Clin Cancer Res; 22(14); 3630-42. ©2016 AACRSee related commentary by Datar and Schalper, p. 3422.
Subject(s)
Adenocarcinoma/pathology , Epithelial-Mesenchymal Transition/physiology , Inflammation/pathology , Lung Neoplasms/pathology , Tumor Microenvironment/physiology , Adenocarcinoma/drug therapy , Adenocarcinoma/metabolism , Adenocarcinoma of Lung , Antineoplastic Agents/therapeutic use , B7 Antigens/metabolism , Biomarkers, Tumor/metabolism , Epithelial-Mesenchymal Transition/drug effects , Humans , Immunotherapy/methods , Inflammation/metabolism , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Mutation/drug effects , Signal Transduction/drug effects , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/metabolism , T-Lymphocytes, Regulatory/pathology , Tumor Microenvironment/drug effectsABSTRACT
Cancer cells modulate the recruitment and function of inflammatory cells to create an immunosuppressive microenvironment that favors tumor growth and metastasis. However, the tumor-derived regulatory programs that promote intratumoral immunosuppression remain poorly defined. Here, we show in a KrasLA1/+p53R172HΔg/+-based mouse model that bone morphogenetic protein-4 (BMP4) augments the expression of the T cell co-inhibitory receptor ligand PD-L1 in the mesenchymal subset of lung cancer cells, leading to profound CD8+ T cell-mediated immunosuppression, producing tumor growth and metastasis. We previously reported in this model that BMP4 functions as a pro-tumorigenic factor regulated by miR-200 via GATA4/6. Thus, BMP4-mediated immunosuppression is part of a larger miR-200-directed gene expression program in tumors that promotes tumor progression, which could have important implications for cancer treatment.
ABSTRACT
Mucin1 (MUC1) promoter has been cloned from the 5' flanking region of the MUC1 gene in breast carcinoma, functionally characterized and applied in gene therapy of breast and esophageal carcinoma. In the present study, we amplified a 786 base pair (bp) MUC1 promoter by two-step nest PCR, and identified the activity and tumor-specificity using an enhanced green fluorescent protein (EGFP) gene as a reporter gene by fluorescence microscopy and flow cytometry analysis in Panc-1, primary normal pancreatic (PNPC), and cervical cancer HeLa cell lines. Subsequently, the human somatostatin receptor subtype 2 (hSSTR2) gene driven by MUC1 promoter was cloned into the pAdTrack to produce recombinant adenovirus AdMUC1-hSSTR2. The anticancer effect of AdMUC1-hSSTR2 was determined in Panc-1. The results demonstrated that there was no AdMUC1-hSSTR2-induced apoptosis, but a significant cell proliferation inhibition even without somatostatin (SST) analogue Octreotide, involved in the up-regulation of the cyclin-dependent kinase (CDK) inhibitors p21 and p27. Moreover, the anticancer effect could not be augmented by the addition of Octreotide, revealing a mechanism that was independent from induction of Octreotide. Therefore, this adenovirus system can be used as a novel, potent and specific tool for gene-targeting therapy in the MUC1 positive pancreatic carcinoma as shown in Panc-1.
Subject(s)
Antigens/genetics , Genetic Therapy , Glycoproteins/genetics , Pancreatic Neoplasms/therapy , Receptors, Somatostatin/genetics , Adenoviridae , Antigens, Neoplasm , Base Sequence , Cytomegalovirus , Genetic Vectors , HeLa Cells , Humans , Molecular Sequence Data , Mucin-1 , Mucins , Pancreatic Neoplasms/genetics , Promoter Regions, Genetic , Receptors, Somatostatin/metabolismABSTRACT
AIM: To investigate the in vitro antitumor effect of adenovirus-mediated small interfering RNAs (siRNAs) on pancreatic cancer and the associated mechanism. METHODS: A 63-nucleotide (nt) oligonucleotide encoding K-ras(val12) and specific siRNA were introduced into pSilencer 3.1-H1, then the H1-RNA promoter and siRNA coding insert were subcloned into pAdTrack to get plasmid pAdTrackH1-K-ras(val12). After homologous recombination in bacteria and transfections of such plasmids into a mammalian packaging cell line 293, siRNA expressing adenovirus AdH1-K-ras(val12) was obtained. Stable suppression of K-ras(val12) was detected by Northern blot and Western blot. Apoptosis in Panc-1 cells was detected by flow cytometry. RESULTS: We obtained adenovirus AdH1-K-ras(val12) carrying the pSilencer 3.1-H1 cassette, which could mediate gene silencing. Through siRNA targeted K-ras(val12), the oncogenic phenotype of cancer cells was reversed. Flow cytometry showed that apoptotic index of Panc-1 cells was significantly higher in the AdH1-K-ras(val12)-treatment group (18.70% at 72 h post-infection, 49.55% at 96 h post-infection) compared to the control groups (3.47%, 3.98% at 72 and 96 h post-infection of AdH1-empty, respectively; 4.21%, 3.78% at 72 and 96 h post-infection of AdH1-p53, respectively) (P<0.05). CONCLUSION: These results demonstrate that adenoviral vectors can be used to mediate RNA interference (RNAi) to induce persistent loss of functional phenotypes. In gene therapy, the selective down-regulation of only the mutant version of a gene allows for highly specific effects on tumor cells, while leaving the normal cells untouched. In addition, the apoptosis of pancreatic cancer cell line Panc-1 can be induced after AdH1-K-ras(val12) infection. This kind of adenovirus based on RNAi might be a promising vector for cancer therapy.
Subject(s)
Gene Silencing , Genes, ras/genetics , Genetic Therapy/methods , Pancreatic Neoplasms/genetics , RNA, Small Interfering/genetics , Adenoviridae/genetics , Base Sequence , Flow Cytometry , Gene Expression Regulation, Neoplastic , HeLa Cells , Humans , In Vitro Techniques , Molecular Sequence Data , Phenotype , Plasmids/geneticsABSTRACT
Epithelial-mesenchymal transition and immunosuppression are crucial for cancer metastasis and treatment resistance. The mechanism by which these distinct processes are co-opted remains incompletely understood. Our recent work has exposed the "dirty affairs" of the 2 at the tumor site, thus calling for a combined therapy to break such a dangerous liaison.
ABSTRACT
Epithelial-mesenchymal transition (EMT) is associated with the metastasis and poor prognosis of cervical cancer. However, the underlying mechanisms are poorly defined. In the present study, we investigated whether Twist plays a direct role in human cervical cancer using immunohistochemical and western blot analyses. Immunohistochemical analysis revealed that Twist is highly expressed in cervical cancer, which correlates with poor tumor pathological differentiation or lymph node metastasis (P<0.05). Depletion of Twist by stable shRNA-mediated knockdown decreased the migratory ability of cancer cell lines in vitro. Suppression or overexpression of Twist also resulted in an altered expression of the molecular mediators of EMT. Furthermore, exogenous TGF-ß promoted EMT by upregulating the expression of Twist through the TGF-ß/Smad3 pathway, and this effect was eliminated by Twist depletion in cancer cells as demonstrated in the in vitro study. The use of in vivo models revealed a decreased tumor proliferation potential in Twist-depleted cancer cells. The results suggested a novel function for Twist in the promotion of EMT via TGF-ß/Smad3 signaling pathway. Thus, Twist constitutes a potential therapeutic target in human cervical cancer.
Subject(s)
Smad3 Protein/genetics , Transforming Growth Factor beta/metabolism , Twist-Related Protein 1/genetics , Uterine Cervical Neoplasms/genetics , Carcinogenesis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Epithelial-Mesenchymal Transition/genetics , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Signal Transduction/drug effects , Smad3 Protein/metabolism , Transforming Growth Factor beta/administration & dosage , Transforming Growth Factor beta/genetics , Twist-Related Protein 1/antagonists & inhibitors , Twist-Related Protein 1/biosynthesis , Uterine Cervical Neoplasms/pathologyABSTRACT
Epidemiological evidence suggests that elevated androgen levels and genetic variation related to the androgen receptor (AR) increase the risk of endometrial cancer (EC). However, the role of AR in EC is poorly understood. We report that two members of the histone demethylase KDM4 family act as major regulators of AR transcriptional activityin EC. In the MFE-296 cell line, KDM4B and AR upregulate c-myc expression, while in AN3CA cells KDM4A and AR downregulate p27kip1. Additionally, KDM4B expression is positively correlated with AR expression in EC cell lines with high baseline AR expression, while KDM4A and AR expression are positively correlated in low-AR cell lines. In clinical specimens, both KDM4B and KDM4A expression are significantly higher in EC tissues than that in normal endometrium. Finally, patients with alterations in AR, KDM4B, KDM4A, and c-myc have poor overall and disease-free survival rates. Together, these findings demonstrate that KDM4B and KDM4A promote EC progression by regulating AR activity.