ABSTRACT
The epidermal growth factor receptor (EGFR) is an important target for cancer therapies. Many head and neck cancer (HNC) cells have been reported to overexpress EGFR; therefore, anti-EGFR therapies have been attempted in patients with HNC. However, its clinical efficacy is limited owing to the development of drug resistance. In this study, we developed an EGFR-targeting immunotoxin consisting of a clinically proven anti-EGFR IgG (cetuximab; CTX) and a toxin fragment (LR-LO10) derived from Pseudomonas exotoxin A (PE) using a novel site-specific conjugation technology (peptide-directed photo-crosslinking reaction), as an alternative option. The immunotoxin (CTX-LR-LO10) showed specific binding to EGFR and properties of a typical IgG, such as stability, interactions with receptors of immune cells, and pharmacokinetics, and inhibited protein synthesis via modification of elongation factor-2. Treatment of EGFR-positive HNC cells with the immunotoxin resulted in apoptotic cell death and the inhibition of cell migration and invasion. The efficacy of CTX-LR-LO10 was evaluated in xenograft mouse models, and the immunotoxin exhibited much stronger tumor suppression than CTX or LR-LO10. Transcriptome analyses revealed that the immunotoxins elicited immune responses and altered the expression of genes related to its mechanisms of action. These results support the notion that CTX-LR-LO10 may serve as a new therapeutic agent targeting EGFR-positive cancers.
Subject(s)
ADP Ribose Transferases , ErbB Receptors , Exotoxins , Head and Neck Neoplasms , Immunoglobulin G , Immunotoxins , Pseudomonas aeruginosa Exotoxin A , Virulence Factors , Humans , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/metabolism , ErbB Receptors/immunology , Animals , Immunotoxins/pharmacology , Head and Neck Neoplasms/drug therapy , Head and Neck Neoplasms/immunology , Head and Neck Neoplasms/metabolism , Mice , Immunoglobulin G/pharmacology , Cell Line, Tumor , Exotoxins/pharmacology , Xenograft Model Antitumor Assays , Cetuximab/pharmacology , Mice, Nude , Bacterial Toxins , Apoptosis/drug effects , Mice, Inbred BALB C , Female , Cell Movement/drug effects , Antineoplastic Agents/pharmacologyABSTRACT
Immune checkpoint blockers (ICBs) targeting programmed cell death protein 1 (PD-1) have been proven to be an effective first-line therapy against programmed cell death 1 ligand 1 (PD-L1; also known as CD274 molecule)-expressing head and neck squamous cell carcinoma (HNSCC) in recent KEYNOTE-048 trial. However, associated changes in the tumor microenvironment (TME) and underlying mechanisms remain elusive. Oral tumors in C57/BL6 mice were induced by administering 7,12-dimethylbenzanthracene into the buccal mucosa. Single-cell suspension was isolated from tumor tissue; proliferating cells were injected subcutaneously into the left flank of mice to establish Ajou oral cancer (AOC) cell lines. Subsequently, a syngeneic PD-L1-expressing HNSCC model was developed by injecting AOC cells into the buccal or tongue area. The model recapitulated human HNSCC molecular features and showed reliable in vivo tumorigenicity with significant PD-L1 expression. ICB monotherapy induced global changes in the TME, including vascular normalization. Furthermore, the antitumor effect of ICB monotherapy was superior to those of other therapeutic agents, including cisplatin and inhibitors of vascular endothelial growth factor receptor 2 (VEGFR2). The ICB-induced antitumorigenicity and TME normalization were alleviated by blocking the type I interferon pathway. In summary, ICB monotherapy is sufficient to induce TME normalization in the syngeneic model; the type I interferon pathway is indispensable in realizing the effects of ICBs. Furthermore, these results explain the underlying mechanism of the efficacy of ICB monotherapy against PD-L1-expressing HNSCC in the KEYNOTE-048 trial.
Subject(s)
B7-H1 Antigen , Head and Neck Neoplasms , Immune Checkpoint Inhibitors , Interferon Type I , Mice, Inbred C57BL , Tumor Microenvironment , Animals , B7-H1 Antigen/metabolism , Immune Checkpoint Inhibitors/pharmacology , Immune Checkpoint Inhibitors/therapeutic use , Tumor Microenvironment/drug effects , Mice , Head and Neck Neoplasms/pathology , Head and Neck Neoplasms/drug therapy , Head and Neck Neoplasms/metabolism , Humans , Cell Line, Tumor , Interferon Type I/metabolism , Squamous Cell Carcinoma of Head and Neck/drug therapy , Squamous Cell Carcinoma of Head and Neck/metabolism , Squamous Cell Carcinoma of Head and Neck/pathology , Female , Signal Transduction/drug effectsABSTRACT
Inflammasomes are multiprotein complexes involved in the host immune response to pathogen infections. Thus, inflammasomes participate in many conditions, such as acne. Recently, it was shown that NETosis, a type of neutrophil cell death, is induced by bacterial infection and is involved in inflammatory diseases such as delayed wound healing in patients with diabetes. However, the relationship between inflammasomes and NETosis in the pathogenesis of inflammatory diseases has not been well studied. In this study, we determined whether NETosis is induced in P. acnes-induced skin inflammation and whether activation of the nucleotide-binding domain, leucine-rich family, and pyrin domain-containing-3 (NLRP3) inflammasome is one of the key factors involved in NETosis induction in a mouse model of acne skin inflammation. We found that NETosis was induced in P. acnes-induced skin inflammation in mice and that inhibition of NETosis ameliorated P. acnes-induced skin inflammation. In addition, our results demonstrated that inhibiting inflammasome activation could suppress NETosis induction in mouse skin. These results indicate that inflammasomes and NETosis can interact with each other to induce P. acnes-induced skin inflammation and suggest that targeting NETosis could be a potential treatment for inflammasome-mediated diseases as well as NETosis-related diseases.
Subject(s)
Acne Vulgaris , Extracellular Traps , Inflammasomes , Inflammation , NLR Family, Pyrin Domain-Containing 3 Protein , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Animals , Inflammasomes/metabolism , Extracellular Traps/metabolism , Extracellular Traps/immunology , Acne Vulgaris/immunology , Mice , Inflammation/immunology , Inflammation/pathology , Skin/pathology , Skin/immunology , Neutrophils/immunology , Neutrophils/metabolism , Mice, Inbred C57BL , Humans , Disease Models, AnimalABSTRACT
Background: Studies on functional roles of BACH1 reveal that BACH1 promotes cancer metastasis and regulates metabolic networks for metastatic processes. However, little is known about BACH1 protein expression in breast tumors and its relevance to clinical variables as a biomarker for patients with breast tumors. Methods: Using a tissue microarray (TMA) of breast tumor tissues isolated from a patient cohort (N = 130) expression of BACH1 and its target gene MCT1 (encoded by SLC16A1) were monitored by immunohistochemistry (IHC) assays and scored for further analyses. We examined the association between scores of BACH1 (Allredscoretotal) or MCT1 (Hscoretotal3×2×1x) with clinical variables including: breast cancer subtypes, tissue types, tumor size, patient's racial/ethnic background, and age group. Groups were compared using the Mann-Whitney U test (or the non-parametric Kruskal-Wallis test when appropriate) for numerical data. A proportional odds ordinal logistic model was used to examine multiple covariates. Associations between variables were evaluated with the Spearman's correlation coefficient. Results: BACH1 and MCT1 expression were detected in 90.76% (N = 118/130) and 92.30% (N = 120/130) of patients by IHC, respectively, in our study. After dichotomizing tumor size (small: 3-25 in diameter vs. big: 27-85 mm in diameter), BACH1 expression scores were significantly higher (p = 0.015) in the bigger tumor group (mean [SD]; 4.20 [1.796]) compared with the smaller tumor group (3.920 [1.693]). Of interest, we also observed significantly higher BACH1 scores (p = 0.004) in tumors from Black women (3.971 [1.514]; N = 69) compared with those of White women (3.02 [1.942]; N = 49). Consistent with mRNA expression analysis, BACH1 expression is most abundant in the basal-like tumors among all subtypes, specifically in Black women, whereas MCT1 expression scores are considerably higher in the basal-like tumors regardless of race. In addition, there was a positive association between BACH1 and MCT1 IHC scores in tumors from Black women, although a weak association between them in tumors from White women. In general, we did not detect associations between MCT1 IHC scores and race, tumor size, tissue types, or patient's age. Conclusions: We found strong associations of BACH1 expression with tumor size and the basal-like subtype, respectively. Importantly, BACH1 expresses significantly higher in tumors from Black women than White women, as well as in the basal-like subtype of breast tumors from Black women. Our study suggests that BACH1 expression could serve as a potential race-associated biomarker indicating poor prognosis.
ABSTRACT
Head and neck squamous cell carcinoma (HNSCC) undergoes stepwise progression from normal tissues to precancerous leukoplakia, primary HNSCC, and metastasized tumors. To delineate the heterogeneity of tumor cells and their interactions during the progression of HNSCC, we employ single-cell RNA-seq profiling for normal to metastasized tumors. We can identify the carcinoma in situ cells in leukoplakia lesions that are not detected by pathological examination. In addition, we identify the cell type subsets of the Galectin 7B (LGALS7B)-expressing malignant cells and CXCL8-expressing fibroblasts, demonstrating that their abundance in tumor tissue is associated with unfavorable prognostic outcomes. We also demonstrate the interdependent ligand-receptor interaction of COL1A1 and CD44 between fibroblasts and malignant cells, facilitating HNSCC progression. Furthermore, we report that the regulatory T cells in leukoplakia and HNSCC tissues express LAIR2, providing a favorable environment for tumor growth. Taken together, our results update the pathobiological insights into cell-cell interactions during the stepwise progression of HNSCCs.
Subject(s)
Head and Neck Neoplasms , Humans , Squamous Cell Carcinoma of Head and Neck , Gene Expression Profiling , LeukoplakiaABSTRACT
Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancer, causing considerable mortality and morbidity worldwide. Although HNSCC management has been extensively studied, the treatment outcomes have not improved - the 5-year survival rate of patients with HNSCC is 40%. Recent studies on the development of a novel HNSCC treatment have highlighted proto-oncogene tyrosine-protein kinase Src (c-Src) as one of the major therapeutic targets. However, the clinical efficacy of c-Src inhibitors against HNSCC was not comparable to that obtained in vitro. Furthermore, the molecular mechanisms underlying the efficacy of c-Src inhibitors remain elusive. In this study, we assessed the efficacy of 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d] pyrimidine (PP2), a selective c-Src inhibitor on HSNCC. Nine HNSCC cell lines (SNU1041, Fraud, SNU46, SNU1076, SNU899, SCC1483, YD15, YD9, and YD10-) were screened, and the effects of PP2 were evaluated using wound healing, apoptosis, and invasion assays. Western blot analysis of downstream markers was conducted to assess the specific mechanism of action of PP2 in HNSCC. The therapeutic efficacy of PP2 was further evaluated in xenograft mice. PP2 reduced tumor cell growth both in vitro and in vivo. Furthermore, it enhanced tumor cell apoptosis in cell lines and prevented metastasis in mice. PP2 also regulated the epithelial-mesenchymal transition pathway downstream of c-Src. More specifically, in SCC1483 and YD15PP2 HNSCC cell lines, PP2 exposure downregulated Erk, Akt/Slug, and Snail but upregulated E-cadherin. These results suggest that PP2 inhibits cell growth and progression in HNSCC by regulating the epithelial-mesenchymal transition pathway.
Subject(s)
Epithelial-Mesenchymal Transition , Head and Neck Neoplasms , Humans , Animals , Mice , Head and Neck Neoplasms/drug therapy , Squamous Cell Carcinoma of Head and Neck/drug therapy , Cell Line, Tumor , Cell ProliferationABSTRACT
Raf Kinase Inhibitory Protein (RKIP) maintains cellular robustness and prevents the progression of diseases such as cancer and heart disease by regulating key kinase cascades including MAP kinase and protein kinase A (PKA). Phosphorylation of RKIP at S153 by Protein Kinase C (PKC) triggers a switch from inhibition of Raf to inhibition of the G protein coupled receptor kinase 2 (GRK2), enhancing signaling by the ß-adrenergic receptor (ß-AR) that activates PKA. Here we report that PKA-phosphorylated RKIP promotes ß-AR-activated PKA signaling. Using biochemical, genetic, and biophysical approaches, we show that PKA phosphorylates RKIP at S51, increasing S153 phosphorylation by PKC and thereby triggering feedback activation of PKA. The S51V mutation blocks the ability of RKIP to activate PKA in prostate cancer cells and to induce contraction in primary cardiac myocytes in response to the ß-AR activator isoproterenol, illustrating the functional importance of this positive feedback circuit. As previously shown for other kinases, phosphorylation of RKIP at S51 by PKA is enhanced upon RKIP destabilization by the P74L mutation. These results suggest that PKA phosphorylation at S51 may lead to allosteric changes associated with a higher-energy RKIP state that potentiates phosphorylation of RKIP at other key sites. This allosteric regulatory mechanism may have therapeutic potential for regulating PKA signaling in disease states.
Subject(s)
Cyclic AMP-Dependent Protein Kinases , Phosphatidylethanolamine Binding Protein , Cyclic AMP-Dependent Protein Kinases/metabolism , Feedback, Physiological , Humans , Male , PC-3 Cells , Phosphatidylethanolamine Binding Protein/genetics , Phosphatidylethanolamine Binding Protein/metabolism , Phosphorylation , Prostatic Neoplasms/metabolism , Protein Kinase C/metabolism , Signal TransductionABSTRACT
The oncogenic expression or mutation of tumor suppressors drives metabolic alteration, causing cancer cells to utilize diverse nutrients. Lactate is a known substrate for cancer cells, yet the regulatory mechanisms of lactate catabolism are limited. Here, we show that a heme-binding transcription factor, BACH1, negatively regulates lactate catabolic pathways in triple-negative breast cancer (TNBC) cells. BACH1 suppresses the transcriptional expression of monocarboxylate transporter 1 (MCT1) and lactate dehydrogenase B, inhibiting lactate-mediated mitochondrial metabolism. In our studies, the depletion of BACH1 either genetically or pharmacologically increased the lactate use of TNBC cells, increasing their sensitivity to MCT1 inhibition. Thus, small inhibitory molecules (SR13800 and AZD3965) blocking MCT1 better suppressed the growth of BACH1-depleted TNBC cells than did the controls. Particularly, hemin treatment degrading BACH1 proteins induced lactate catabolism in TNBC cells, generating synthetic lethality with MCT1 inhibition. Our data indicates that targeting BACH1 generates metabolic vulnerability and increases sensitivity to lactate transporter inhibition, suggesting a potential novel combination therapy for cancer patients with TNBC.
Subject(s)
Basic-Leucine Zipper Transcription Factors , Lactic Acid , Triple Negative Breast Neoplasms , Basic-Leucine Zipper Transcription Factors/metabolism , Heme/metabolism , Humans , Lactic Acid/metabolism , Monocarboxylic Acid Transporters/genetics , Monocarboxylic Acid Transporters/metabolism , Transcription Factors/metabolism , Triple Negative Breast Neoplasms/metabolismABSTRACT
Melanoma, the most serious type of skin cancer, exhibits a high risk of metastasis. Although chemotherapeutic treatment for metastatic melanoma improves disease outcome and patient survival, some patients exhibit resistance or toxicity to the drug treatment regime. OTUB1 is a deubiquitinating enzyme overexpressed in several cancers. In this study, we investigated the effects of inhibiting OTUB1 expression on melanoma-cell proliferation and viability and identified the underlying molecular mechanism of action of OTUB1. We did endogenous OTUB1 knockdown in melanoma cells using short interfering RNA, and assessed the resulting phenotypes via MTT assays, Western blotting, and cell-cycle analysis. We identified differentially expressed genes between OTUB1-knockdown cells and control cells using RNA sequencing and confirmed them via Western blotting and reverse transcription polymerase chain reaction. Furthermore, we investigated the involvement of apoptotic and cell survival signaling pathways upon OTUB1 depletion. OTUB1 depletion in melanoma cells decreased cell viability and caused simultaneous accumulation of cells in the sub-G1 phase, indicating an increase in the apoptotic-cell population. RNA sequencing of OTUB1-knockdown cells revealed an increase in the levels of the apoptosis-inducing protein TRAIL. Additionally, OTUB1-knockdown cells exhibited increased sensitivity to PLX4032, a BRAF inhibitor, implying that OTUB1 and BRAF act collectively in regulating apoptosis. Taken together, our findings show that OTUB1 induces apoptosis of melanoma cells in vitro, likely by upregulating TRAIL, and suggest that approaches targeting OTUB1 can be developed to provide novel therapeutic strategies for treating melanoma. [BMB Reports 2021; 54(12): 608-613].
Subject(s)
Apoptosis , Deubiquitinating Enzymes , Melanoma , TNF-Related Apoptosis-Inducing Ligand , Apoptosis/genetics , Apoptosis Regulatory Proteins , Cell Line, Tumor , Deubiquitinating Enzymes/genetics , Deubiquitinating Enzymes/metabolism , Gene Knockdown Techniques , Humans , Melanoma/drug therapy , Melanoma/genetics , Melanoma/pathology , TNF-Related Apoptosis-Inducing Ligand/genetics , TNF-Related Apoptosis-Inducing Ligand/metabolism , TNF-Related Apoptosis-Inducing Ligand/pharmacologyABSTRACT
Head and neck squamous cell carcinoma (HNSCC) metastasizes to the locoregional lymph nodes at high rates and is related to poor clinical outcomes. However, the mechanism by which cancer cells migrate to the lymph nodes is unclear. To address this, we established a conditioned medium culture system for HNSCC cells and lymphatic endothelial cells (LECs) and investigated their crosstalk. Stimulation with tumor-conditioned medium (TCM) activated LECs, resulting in a robust increase in cell proliferation to induce lymphatic hyperplasia. Further, stimulation of HNSCC cells with activated LEC Conditioned media (TCM-LEC CM) induced cell invasion. Among various chemokines, CXCL5 promoted the invasion of TCM-LEC CM-treated HNSCC cells. The level of CXCL5 protein was higher in cancer tissues than those in normal tissues from HNSCC patients. Furthermore, treatment with SB225002, a CXCR2 (CXCL5 receptor) inhibitor, resulted in decreased lymph node metastasis in vivo. In conclusion, inhibition of CXCL5-CXCR2 signaling between cancer cells and LECs suppresses cancer cell invasion and metastasis in vitro and in vivo. This novel therapeutic strategy might be a practical approach to the clinical management of HNSCC.
Subject(s)
Cell Communication , Chemokine CXCL5/metabolism , Endothelial Cells/metabolism , Head and Neck Neoplasms/metabolism , Neoplasm Proteins/metabolism , Receptors, Interleukin-8B/metabolism , Signal Transduction , Animals , Cell Line, Tumor , Chemokine CXCL5/genetics , Endothelial Cells/pathology , Female , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/pathology , Humans , Male , Mice , Mice, Nude , Neoplasm Metastasis , Neoplasm Proteins/genetics , Receptors, Interleukin-8B/geneticsABSTRACT
Epithelial specific ETS-1 (ESE-1) belongs to the E26 transformation-specific transcription factor superfamily and is of great interest as a potential target for managing several types of cancer. Despite its clinical significance, the documented effects of ESE-1 on cancer development and progression are contradictory and its underlying biological mechanism of action remains elusive. The objectives of this study are to investigate whether ESE-1 is a tumor suppressor and to identify dietary anti-cancer compound to activate ESE-1 expression in human colon cancer model. ESE-1 knockout and xenograft mouse models were used to examine the effect of ESE-1 in colon tumorigenesis. Stable human colon cancer cell lines were used for in vitro mechanistic studies. ESE-1 knockout in mice increased azoxymethane (AOM)-induced and dextran sulfate sodium (DSS)-promoted formation of aberrant crypt foci (ACF). Conversely, overexpression of ESE-1 suppressed tumorigenicity in a xenograft mouse study, and repressed anchorage-independent growth and migration/invasion in human colon cancer cells. Full length ESE-1 localized abundantly in the nucleus, and internal deletion of nuclear localization sequence 2 (NLS2) reduced nuclear ESE-1. Three lysine residues (318 KKK320 ) in the NLS2 determine its nuclear localization. We identified epigallocatechin-3-gallate (EGCG) that acts as a transcriptional activator of ESE-1 in human colon cancer cells. These findings propose a novel and promising molecular target of dietary anti-cancer compounds for prevention of colon cancer.
Subject(s)
Antineoplastic Agents, Phytogenic/administration & dosage , Catechin/analogs & derivatives , Colonic Neoplasms/drug therapy , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Proto-Oncogene Proteins c-ets/genetics , Proto-Oncogene Proteins c-ets/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Animals , Antineoplastic Agents, Phytogenic/pharmacology , Azoxymethane/adverse effects , Caco-2 Cells , Catechin/administration & dosage , Catechin/pharmacology , Cell Line, Tumor , Cell Movement/drug effects , Cell Nucleus/metabolism , Colonic Neoplasms/chemically induced , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , DNA-Binding Proteins/chemistry , Dextran Sulfate/adverse effects , Gene Expression Regulation, Neoplastic/drug effects , Gene Knockdown Techniques , HCT116 Cells , HT29 Cells , Humans , Mice , Nuclear Localization Signals , Proto-Oncogene Proteins c-ets/chemistry , Transcription Factors/chemistry , Xenograft Model Antitumor AssaysABSTRACT
Although TRAIL can directly induce cell death in some cancer cells, it appears that TRAIL resistance exists in many cancers. This study focuses on anti-cancer drugs for TRAIL-resistant head and neck cancer (HNC) to provide further progress toward effective cancer therapy. Results indicate in TRAIL-resistant HNC cells, that combined TRAIL and VPA treatment greatly reduced cell viability and therefore induced cell death, relative to treatment with TRAIL or VPA alone. A caspase-dependent signaling pathway was demonstrated, and combined treatment with TRAIL and VPA also significantly decreased the expression of HDAC4. When we pretreated cells with z-VAD followed by combined treatment with TRAIL and VPA, cell death was blocked with no reduction in expression of HDAC4. To confirm that cell death involved HDAC4 in HNC cells, we knocked down expression of HDAC4 with siRNA, followed by treatment with TRAIL and VPA. Results showed that loss of HDAC4 sensitized the TRAIL-resistant HNC cells to apoptotic cell death. Finally, we showed elevated expression of HDAC4 in HNC tissues compared to normal tissues obtained from the same patients. In conclusion, we suggest that combined VPA and TRAIL treatment may be a promising therapy for HNC via HDAC4 degradation.
Subject(s)
Drug Resistance, Neoplasm/drug effects , Head and Neck Neoplasms/drug therapy , Histone Deacetylases/chemistry , Repressor Proteins/chemistry , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Valproic Acid/pharmacology , Caspases/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Drug Synergism , Gene Expression Regulation, Neoplastic/drug effects , Gene Knockdown Techniques , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/metabolism , Histone Deacetylases/genetics , Humans , Proteolysis , Repressor Proteins/genetics , Signal Transduction/drug effects , Up-Regulation/drug effectsABSTRACT
Lung cancer is the first leading cause of cancerrelated death in the United States. Nonsmall cell lung cancer (NSCLC) is the most common type of lung cancer and is associated with a poor patient prognosis. Identification of promising molecular targets is required for the effective prevention and therapy of NSCLC. Epithelialspecific ETS1 (ESE1) belongs to the superfamily of ETS transcription factors. The effect of ESE1 on tumorigenesis is controversial in several types of cancer while its role in lung cancer remains unknown. The present study was designed to investigate whether ESE1 expression affects tumorigenic activity using human NSCLC cells and a mouse xenograft model. ESE1 expression suppressed anchorageindependent growth in soft agar assay and led to an increase in G1 arrest and apoptosis in human NSCLC cells. ESE1 expression suppressed the invasion and migration of human NSCLC cells. Western blot analysis, RTPCR and promoter assay indicated that ESE1 expression was transcriptionally downregulated by treatment of transforming growth factor (TGF)ß, an EMT (epithelialmesenchymal transition) stimulator. The xenograft study indicated that ESE1 expression inhibited tumor formation and development. Our data demonstrated that ESE1 plays a key role as a tumor suppressor in human NSCLC.
Subject(s)
Biomarkers, Tumor/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Movement , Cell Proliferation , DNA-Binding Proteins/metabolism , Lung Neoplasms/pathology , Proto-Oncogene Proteins c-ets/metabolism , Transcription Factors/metabolism , Transforming Growth Factor beta/metabolism , Animals , Apoptosis , Carcinogenesis , Carcinoma, Non-Small-Cell Lung/metabolism , Epithelial-Mesenchymal Transition , Humans , Lung Neoplasms/metabolism , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasm Invasiveness , Signal Transduction , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Anoctamin-1 (ANO1) acts as a Ca(2+)-activated Cl(-) channel in various normal tissues, and its expression is increased in several different types of cancer. Therefore, understanding the regulation of ANO1 surface expression is important for determining its physiological and pathophysiological functions. However, the trafficking mechanism of ANO1 remains elusive. Here, we report that segment a (N-terminal 116 amino acids) of ANO1 is crucial for its surface expression, and we identified 14-3-3γ as a binding partner for anterograde trafficking using yeast two-hybrid screening. The surface expression of ANO1 was enhanced by 14-3-3γ, and the Thr9 residue of ANO1 was critical for its interaction with 14-3-3γ. Gene silencing of 14-3-3γ and/or ANO1 demonstrated that suppression of ANO1 surface expression inhibited migration and invasion of glioblastoma cells. These findings provide novel therapeutic implications for glioblastomas, which are associated with poor prognosis.
Subject(s)
14-3-3 Proteins/metabolism , Anoctamin-1/chemistry , Anoctamin-1/metabolism , Brain Neoplasms/metabolism , Glioblastoma/metabolism , Neoplasm Proteins/chemistry , Neoplasm Proteins/metabolism , Binding Sites , Cell Line, Tumor , Cell Membrane/metabolism , Cell Movement , Disease Progression , Gene Expression Regulation, Neoplastic , HEK293 Cells , Humans , Neoplasm Invasiveness , Protein Binding , Two-Hybrid System TechniquesABSTRACT
PURPOSE: Tolfenamic acid (TA), a non-steroidal anti-inflammatory drug, is known to exhibit antitumor effects in various cancers apart from nasopharyngeal cancer (NPC). NPC exhibits high invasiveness, as well as metastatic potential, and patients continue to suffer from residual, recurrent, or metastatic disease even after chemoradiation therapy. Therefore, new treatment strategies are needed for NPC. In this study, we investigated the efficacy and molecular mechanisms of TA in NPC treatment. MATERIALS AND METHODS: TA-induced cell death was detected by cell viability assay in the NPC cell lines, HNE1 and HONE1. Wound healing assay, invasion assay, and Western blot analysis were used to evaluate the antitumor effects of TA in NPC cell lines. RESULTS: Treatment with TA suppressed the migration and invasion of HNE1 and HONE1 cells. Hepatocyte growth factor enhanced the proliferation, migration, and invasion abilities of NPC cells. This enhancement was successfully inhibited by TA treatment. Treatment with TA increased phosphorylation of p38, and the inhibition of p38 with SB203580 reversed the cytotoxic, anti-invasive, and anti-migratory effects of TA treatment in NPC cell lines. Moreover, inhibition of p38 also reversed the decrease in expression of Slug that was induced by TA treatment. CONCLUSION: In conclusion, the activation of p38 plays a role in mediating TA-induced cytotoxicity and inhibition of invasion and migration via down-regulation of Slug.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Hepatocyte Growth Factor/pharmacology , Nasopharyngeal Neoplasms/drug therapy , Neoplasm Invasiveness/prevention & control , ortho-Aminobenzoates/pharmacology , Animals , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Carcinoma , Cell Line, Tumor , Down-Regulation , Gastropoda , Gene Expression Regulation, Neoplastic/drug effects , Hepatocyte Growth Factor/metabolism , Humans , Imidazoles , MAP Kinase Signaling System/drug effects , Nasopharyngeal Carcinoma , Nasopharyngeal Neoplasms/metabolism , Nasopharyngeal Neoplasms/pathology , Phosphorylation/drug effects , Pyridines , ortho-Aminobenzoates/therapeutic useABSTRACT
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) represents an effective agent for the treatment of many cancers, though the majority of thyroid cancers are found to be resistant. Therefore it would be necessary to identify agents capable of increasing the sensitivity of these cancers to TRAIL-mediated cell death. Here, we examined the therapeutic effect and its underlying mechanism of combination treatment of TRAIL and histone deacetylase inhibitor, Valproic acid (VPA) in vitro using human papillary thyroid cancer (PTC) cells and in vivo using an orthotopic mouse model of PTC. TRAIL-VPA combination therapy synergistically induced apoptotic cell death in TRAIL-resistant PTC through caspase activation. In addition, downregulation of antioxidant transcription factor, Nrf2 by co-treatment of TRAIL-VPA induces cell death via suppression of Bcl-xL in vitro and in vivo; these effects were further enhanced following siRNA inhibition of these proteins in combination with TRAIL or TRAIL-VPA. Taken together, VPA sensitized TRAIL-resistant PTC cells to apoptotic cell death through involvement of Nrf2 and Bcl-xL. Thus, the combination of VPA and TRAIL may be a promising therapy for TRAIL-resistant PTC.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/drug effects , Carcinoma/drug therapy , Drug Resistance, Neoplasm , Histone Deacetylase Inhibitors/pharmacology , NF-E2-Related Factor 2/metabolism , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Thyroid Neoplasms/drug therapy , Valproic Acid/pharmacology , bcl-X Protein/metabolism , Animals , Carcinoma/genetics , Carcinoma/metabolism , Carcinoma/pathology , Carcinoma, Papillary , Cell Line, Tumor , Cell Survival/drug effects , Dose-Response Relationship, Drug , Down-Regulation , Drug Synergism , Humans , Male , Mice, Inbred BALB C , Mice, Nude , NF-E2-Related Factor 2/genetics , RNA Interference , Signal Transduction/drug effects , Thyroid Cancer, Papillary , Thyroid Neoplasms/genetics , Thyroid Neoplasms/metabolism , Thyroid Neoplasms/pathology , Time Factors , Transfection , bcl-X Protein/geneticsABSTRACT
PURPOSE: It has been reported that the abnormal activation of receptor tyrosine kinases is associated with the development of many human carcinomas and the high activation of EGFR and Met mediates the tumorigenicity of laryngeal carcinoma. In this study, we have done the therapeutic efficacy of ME22S (a novel EGFR/Met bispecific antibody) in laryngeal carcinoma in vitro and in vivo was thoroughly evaluated. METHODS: The effects of ME22S on cell viability was assessed through MTT assays, and then Western blotting and immunocytochemistry were used to determine the expression of EGFR and Met. Also, wound healing and invasion assays were performed to observe the inhibitory effects of ME22S. RESULTS: We found the ability of ME22S reducing the expression of both EGFR and Met and significantly inhibiting the cell migration, invasion, and proliferation of SNU899 and HN3 in vitro. Also, the notably reduced levels of p-Met, p-ERK, and p-AKT were found when the cells were treated with only ME22S alone or with HGF together. Meanwhile, ME22S, interestingly enough, caused caspase-3-dependent apoptotic cell death when HN3 cells were treated with ME22S for 72 h, decreased the HGF-induced Slug expression, and also inhibited the tumor growth of HN3 cells in a xenograft model in vivo. CONCLUSIONS: Taken together, our findings suggest that the dual inhibition of EGFR and Met through ME22S largely suppresses the invasion and growth of laryngeal carcinoma both in vitro and in vivo, hence, can be a practical approach as a novel therapeutic strategy for the treatment of laryngeal carcinoma.
Subject(s)
Antibodies, Bispecific/pharmacology , Cell Movement/drug effects , Cell Proliferation/drug effects , ErbB Receptors/antagonists & inhibitors , Laryngeal Neoplasms/prevention & control , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Animals , Biomarkers, Tumor/metabolism , Humans , Laryngeal Neoplasms/drug therapy , Laryngeal Neoplasms/pathology , Male , Mice , Mice, Inbred BALB C , Neoplasm Invasiveness , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: The purpose of this study is to determine whether luminacin, a marine microbial extract from the Streptomyces species, has anti-tumor effects on head and neck squamous cell carcinoma (HNSCC) cell lines via autophagic cell death. MATERIALS AND METHODS: Inhibition of cell survival and increased cell death was measured using cell viability, colony forming, and apoptosis assays. Migration and invasion abilities of head and cancer cells were evaluated using wound healing, scattering, and invasion assays. Changes in the signal pathway related to autophagic cell death were investigated. Drug toxicity of luminacin was examined in in vitro HaCaT cells and an in vivo zebrafish model. RESULTS: Luminacin showed potent cytotoxicity in HNSCC cells in cell viability, colony forming, and fluorescence-activated cell sorting analysis. In vitro migration and invasion of HNSCC cells were attenuated by luminacin treatment. Combined with Beclin-1 and LC3B, Luminacin induced autophagic cell death in head and neck cancer cells. In addition, in a zebrafish model and human keratinocyte cell line used for toxicity testing, luminacin treatment with a cytotoxic concentration to HNSCC cells did not cause toxicity. CONCLUSION: Taken together, these results demonstrate that luminacin induces the inhibition of growth and cancer progression via autophagic cell death in HNSCC cell lines, indicating a possible alternative chemotherapeutic approach for treatment of HNSCC.
Subject(s)
Autophagy/drug effects , Benzaldehydes/pharmacology , Benzaldehydes/therapeutic use , Carcinoma, Squamous Cell/pathology , Cell Extracts/pharmacology , Cell Extracts/therapeutic use , Head and Neck Neoplasms/pathology , Spiro Compounds/pharmacology , Spiro Compounds/therapeutic use , Animals , Benzaldehydes/adverse effects , Carcinoma, Squamous Cell/drug therapy , Cell Extracts/adverse effects , Cell Line, Tumor , Cell Movement/drug effects , Cell Survival/drug effects , Disease Progression , Head and Neck Neoplasms/drug therapy , Humans , Keratinocytes/cytology , Keratinocytes/drug effects , Neoplasm Invasiveness/prevention & control , Spiro Compounds/adverse effects , Zebrafish/embryologyABSTRACT
Hepatocyte growth factor (HGF) and its receptor, Met, regulate skeletal muscle differentiation. In the present study, we identified a novel alternatively spliced isoform of Met lacking exon 13 (designated Δ13Met), which is expressed mainly in human skeletal muscle. Alternative splicing yielded a truncated Met having extracellular domain only, suggesting an inhibitory role. Indeed, Δ13Met expression led to a decrease in HGF-induced tyrosine phosphorylation of Met and ERK phosphorylation, as well as cell proliferation and migration via sequestration of HGF. Interestingly, in human primary myoblasts undergoing differentiation, Δ13Met mRNA and protein levels were rapidly increased, concomitantly with a decrease in wild type Met mRNA and protein. Inhibition of Δ13Met with siRNA led to a decreased differentiation, whereas its overexpression potentiated differentiation of human primary myoblasts. Furthermore, in notexin-induced mouse injury model, exogenous Δ13Met expression enhanced regeneration of skeletal muscle, further confirming a stimulatory role of the isoform in muscle cell differentiation. In summary, we identified a novel alternatively spliced inhibitory isoform of Met that stimulates muscle cell differentiation, which confers a new means to control muscle differentiation and/or regeneration.
Subject(s)
Cell Differentiation , Hepatocyte Growth Factor/metabolism , Muscle Fibers, Skeletal/cytology , Proto-Oncogene Proteins c-met/metabolism , Alternative Splicing , Animals , Biopsy , Cell Line , Cell Proliferation , Humans , Mice , Mice, Inbred C57BL , Myoblasts/metabolism , NIH 3T3 Cells , Phosphorylation , Protein Isoforms/metabolism , RNA Interference , Regeneration , Signal TransductionABSTRACT
PURPOSE: Past studies have reported that S-allylcysteine (SAC) inhibits the migration and invasion of cancer cells through the restoration of E-cadherin, the reduction of matrix metalloproteinase (MMP) and Slug protein expression, and inhibition of the production of reactive oxygen species (ROS). Furthermore, evidence is emerging that shows that ROS induced by radiation could increase Met activation. Following on these reports of SAC and Met, we investigated whether SAC could suppress Met activation. MATERIALS AND METHODS: Wound healing, invasion, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT), soft agar colony forming, western blotting, and gelatin zymography assays were performed in the human nasopharyngeal cancer cell lines HNE1 and HONE1 treated with SAC (0, 10, 20, or 40 mM) and hepatocyte growth factor (HGF). RESULTS: This study showed that SAC could suppress the migration and invasion of HNE1 and HONE1 cell lines by inhibiting p-Met. An increase of migration and invasion induced by HGF and its decrease in a dose dependent manner by SAC in wound healing and invasion assays was observed. The reduction of p-Met by SAC was positively correlated with p-focal adhesion kinase (p-FAK) and p-extracellular related kinase (p-ERK in both cell lines). SAC reduced Slug, MMP2, and MMP9 involved in migration and invasion with the inhibition of Met-FAK signaling. CONCLUSION: These results suggest that SAC inhibited not only Met activation but also the downstream FAK, Slug, and MMP expression. Finally, SAC may be a potent anticancer compound for nasopharyngeal cancer treated with radiotherapy.