ABSTRACT
The Hippo pathway plays a pivotal role in tissue homeostasis and tumor suppression. YAP and TAZ are downstream effectors of the Hippo pathway, and their activities are tightly suppressed by phosphorylation-dependent cytoplasmic retention. However, the molecular mechanisms governing YAP/TAZ nuclear localization have not been fully elucidated. Here, we report that Mastermind-like 1 and 2 (MAML1/2) are indispensable for YAP/TAZ nuclear localization and transcriptional activities. Ectopic expression or depletion of MAML1/2 induces nuclear translocation or cytoplasmic retention of YAP/TAZ, respectively. Additionally, mutation of the MAML nuclear localization signal, as well as its YAP/TAZ interacting region, both abolish nuclear localization and transcriptional activity of YAP/TAZ. Importantly, we demonstrate that the level of MAML1 messenger RNA (mRNA) is regulated by microRNA-30c (miR-30c) in a cell-density-dependent manner. In vivo and clinical results suggest that MAML potentiates YAP/TAZ oncogenic function and positively correlates with YAP/TAZ activation in human cancer patients, suggesting pathological relevance in the context of cancer development. Overall, our study not only provides mechanistic insight into the regulation of YAP/TAZ subcellular localization, but it also strongly suggests that the miR30c-MAML-YAP/TAZ axis is a potential therapeutic target for developing novel cancer treatments.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cell Nucleus/metabolism , DNA-Binding Proteins/metabolism , Neoplasms/metabolism , Trans-Activators/metabolism , Transcription Factors/metabolism , Adaptor Proteins, Signal Transducing/genetics , Carcinogenesis/genetics , Carcinogenesis/metabolism , Cell Nucleus/genetics , DNA-Binding Proteins/genetics , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , Neoplasms/genetics , Protein Transport , Signal Transduction , Trans-Activators/genetics , Transcription Factors/genetics , Transcriptional Coactivator with PDZ-Binding Motif Proteins , YAP-Signaling ProteinsABSTRACT
INTRODUCTION: Human epidermal growth factor 2 (HER2) gene overexpression in breast carcinoma cell lines has been shown to drive mammary carcinogenesis and tumor growth and invasion through its effects on mammary stem cells. OBJECTIVE: Therefore, we investigated the mechanism by which HER2 regulates cancer stem cell (CSC) activity in gastric cancer cells. METHODS: HER2 was transfected into MKN28 gastric cancer cells, and its role in regulating CSC activity was determined by characterizing the HER2-overexpressing cells. RESULTS: The sphere formation assay revealed that the sphere sizes and frequency of sphere formation were significantly greater for the HER2-overexpressing cells than for the MKN28 control cells. The CSC markers Oct-4 and BMI1 were more highly expressed in the HER2-overexpressing cells, as were the EMT markers. This was accompanied by a significant enhancement in cellular invasion of the Matrigel and migration. The E-cadherin level was significantly downregulated, and the mesenchymal marker Snail upregulated, in the HER2-transfected cells. HER2 overexpression activated the well-characterized CSC-associated Wnt/ß-catenin signaling pathway, as shown by the luciferase assay. After treatment of these cells with the Wnt signal inhibitor PRI-724, the BMI1 and Oct-4 levels were decreased for 24 h and Snail was also downregulated. Immunofluorescence staining revealed the significant restoration of E-cadherin levels in the HER2-transfected cells after PRI-724 treatment. CONCLUSIONS: These results established a role for HER2 in regulating gastric CSC activity, with Wnt/ß-catenin signaling being mediated via a HER2-dependent pathway. In summary, HER2-overexpressing gastric cancer cells exhibited increased stemness and invasiveness and were regulated by Wnt/ß-catenin signaling.
Subject(s)
Neoplastic Stem Cells/physiology , Receptor, ErbB-2/physiology , Stomach Neoplasms/pathology , Wnt Signaling Pathway/physiology , Antigens, CD/analysis , Cadherins/analysis , Cell Line, Tumor , Humans , Neoplasm Invasiveness , Octamer Transcription Factor-3/analysis , Polycomb Repressive Complex 1/analysis , Receptor, ErbB-2/analysis , Stomach Neoplasms/chemistry , beta Catenin/analysisABSTRACT
BACKGROUND: Anoctamin (ANO)/transmembrane member 16 (TMEM16) proteins mediate diverse physiological and pathophysiological functions including cancer cell proliferation. The present study aimed to identify the role of ANOs in pancreatic cancer. METHODS: In an initial screen of ANOs, ANO9/TMEM16J was overexpressed in pancreatic cancer cells, and its role in the pathogenesis of pancreatic cancer was evaluated using an integrated in vitro and in vivo approach. To determine clinical relevance of the experimental findings, the prognostic value of ANO9 was evaluated in patients with pancreatic cancer. RESULTS: The ANO9 mRNA and protein levels were increased in pancreatic cancer-derived cells. Exogenous expression of ANO9 in PANC-1 cells significantly increased cell proliferation in cell cultures and in mice. In contrast, knockdown of ANO9 in AsPC-1, BxPC-3, and Capan-2 cells strongly inhibited cell proliferation. Mechanistic analysis suggested that physical association of ANO9 with epidermal growth factor receptor (EGFR) underlies ANO9-induced cell proliferation. Knockdown of ANO9 augmented the effects of the EGFR inhibitor and the cytotoxic agent on pancreatic cancer cell proliferation. In addition, high ANO9 expression is a poor prognostic factor in patients with pancreatic cancer. CONCLUSIONS: The ANO9/TMEM16J appears to be a clinically useful prognostic marker for pancreatic cancer and a potential therapeutic target.
Subject(s)
Anoctamins/genetics , Anoctamins/metabolism , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/metabolism , ErbB Receptors/metabolism , Membrane Proteins/metabolism , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Phospholipid Transfer Proteins/genetics , Phospholipid Transfer Proteins/metabolism , Adult , Aged , Aged, 80 and over , Animals , Anti-Bacterial Agents/therapeutic use , Antineoplastic Agents/pharmacology , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Carcinogenesis , Carcinoma, Pancreatic Ductal/drug therapy , Cell Line, Tumor , Cell Proliferation/genetics , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Disease-Free Survival , Doxycycline/therapeutic use , ErbB Receptors/antagonists & inhibitors , Erlotinib Hydrochloride/pharmacology , Female , Gene Knockdown Techniques , HEK293 Cells , Humans , Male , Membrane Proteins/genetics , Mice , Middle Aged , Neoplasm Transplantation , Pancreatic Neoplasms/drug therapy , Prognosis , RNA, Messenger/metabolism , Survival Rate , Tumor Stem Cell Assay , GemcitabineABSTRACT
Bile duct cancer, or cholangiocarcinoma, is a rare disease with limited treatment options that include surgery and cytotoxic chemotherapy. The high recurrence rate and poor prognosis of this type of cancer highlights the need to identify new and more effective therapeutic targets. In this study, we found that AXL, a receptor tyrosine kinase, is highly expressed in biliary cancer patients and significantly correlated with poor patient outcomes, including metastasis and low survival rates. We also demonstrated that targeting AXL inhibits tumor progression. In vitro studies with bile duct cancer cells (SNU1196 and HUCCT1) showed that genetic knockdown of AXL significantly reduced both tumor cell growth and invasion. In addition, in vivo studies using subcutaneous and orthotopic intrahepatic models demonstrated that genetic inhibition of AXL resulted in tumor-growth delay. To further examine the possible clinical translation of AXL inhibition in the clinic, we tested the efficacy of AVB-500, a soluble AXL receptor, in reducing AXL activation and tumor growth. AVB-500 was effective at inhibiting AXL activation and decreasing the growth and invasion of SNU1196 and HUCCT1 tumors which possess high AXL expression. Most importantly, AVB-500 was highly effective at decreasing tumor dissemination of bile duct tumor cells in the peritoneal cavity. This study strongly supports the idea of using the AXL receptor as a new therapeutic target to treat the growth and progression of biliary cancer.
ABSTRACT
To characterize the radiation-enhancing effects on human cancer cells and underlying mechanisms of celecoxib, a cyclooxygenase (COX)-2 selective inhibitor, and to ascertain whether its effects are COX-2 dependent. Clonogenic cytotoxicity assays and radiation survival assays after treatment with celecoxib +/- radiation were done on four human cancer cell lines that expressed differential COX-2 levels. Stably COX-2 knocked down or overexpressed cell lines were developed, and clonogenic assays, apoptosis assays, or cell cycle change measurements were conducted after treatment with celecoxib +/- radiation. Prostaglandin E(2) (PGE2) was applied to medium after treatment with celecoxib +/- radiation to determine whether the radiation-enhancing effect associated with celecoxib results from reduced generation of prostaglandin. Celecoxib's radiation-enhancing effect was observed in COX-2-expressing A549 and NCI-H460 cells but was not observed in the COX-2 nonexpressing MCF-7 and HCT-116 cells. Celecoxib's radiation-enhancing effects in A549 cells were shown to disappear after the administration of COX-2 knocked down. In contrast, the HCT-116 cells were radiosensitized by celecoxib after being transfected with COX-2 expression vector. The addition of PGE2 after treatment with celecoxib +/- radiation had no significant effects on celecoxib's radiation-enhancing effects in A549 and COX-2 transfected HCT-116 cells. Radiation-induced G2-M arrest was enhanced and sustained in the COX-2-overexpressing cells compared with that seen in COX-2 low-expressing cells. Celecoxib or NS-398 effected no changes or attenuated radiation-induced G(2)-M arrest in the COX-2-overexpressing cells but further enhanced the radiation-induced G(2)-M arrest in the COX-2 low-expressing cells. Celecoxib's radiation-enhancing effects seem to occur in a COX-2 expression-dependent manner in the cancer cells. This effect does not seem to be the result of reduced PGE2 generation. Celecoxib may exert an inhibitory effect on enhanced radiation-induced G2-M arrest in the COX-2-overexpressing cells, which may allow the arrested cells to enter mitosis and die after radiation, but may also further enhance radiation-induced G2-M arrest in the COX-2 low-expressing cells, by virtue of another mechanism.
Subject(s)
Cyclooxygenase 2 Inhibitors/pharmacology , Neoplasms/enzymology , Neoplasms/radiotherapy , Pyrazoles/pharmacology , Radiation-Sensitizing Agents/pharmacology , Sulfonamides/pharmacology , Apoptosis/drug effects , Apoptosis/radiation effects , Celecoxib , Cell Cycle/physiology , Cell Cycle/radiation effects , Cell Line, Tumor , Combined Modality Therapy , Cyclooxygenase 2/biosynthesis , Cyclooxygenase 2/metabolism , Dinoprostone/pharmacology , HCT116 Cells , Humans , Neoplasms/drug therapy , Radiation Tolerance/drug effectsABSTRACT
PURPOSE: This study was conducted to evaluate the biological features of murine hepatocarcinoma according to different tumor microenvironmental models and to determine the change in molecular and immunologic responses after radiation. MATERIALS AND METHODS: Tumor models were established in the liver (orthotopic) and thigh (heterotopic) of male C3H/HeN mice. Tumor growth and lung metastasis were assessed in these models. To evaluate the radiation effect, the tumors were irradiated with 10 Gy. Factors associated with tumor microenvironment including vascular endothelial growth factor (VEGF), cyclooxygenase-2 (COX-2), transforming growth factor beta1 (TGF-ß1), CD31, and serum interleukin-6 (IL-6) were evaluated. Tumor-infiltrating regulatory immune cells, regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSCs) were also analyzed. RESULTS: A higher number of lung metastases were observed in the orthotopic tumor model than in the heterotopic tumor model. VEGF, CD31, COX-2, and TGF-ß1 expression was more prominent in the orthotopic tumor model than in the heterotopic tumor model. Expression of the angiogenic factor VEGF and key regulatory molecules (TGF-ß1 and COX-2) decreased following radiation in the orthotopic tumor model, while the serum IL-6 level increased after radiation. In the orthotopic tumor model, the number of both Tregs and MDSCs in the tumor burden decreased after radiation. CONCLUSION: The orthotopic tumor model showed higher metastatic potential and more aggressive molecular features than the heterotopic tumor model. These findings suggest that the orthotopic tumor mouse model may be more reflective of the tumor microenvironment and suitable for use in the translational research of radiation treatment.
Subject(s)
Liver Neoplasms/pathology , Liver Neoplasms/radiotherapy , Tumor Microenvironment , Animals , Cell Line, Tumor , Cyclooxygenase 2/metabolism , Interleukin-6/blood , Liver Neoplasms/immunology , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/radiation effects , Male , Mice , Mice, Inbred C3H , Myeloid-Derived Suppressor Cells/immunology , Myeloid-Derived Suppressor Cells/radiation effects , Neoplasm Transplantation , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/radiation effects , Transforming Growth Factor beta1/metabolism , Tumor Microenvironment/radiation effects , Vascular Endothelial Growth Factor A/metabolismABSTRACT
BACKGROUND: Radiotherapy (RT) is one of the major non-operative treatment modalities for treating lung cancer. Tomotherapy is an advanced type of intensity-modulated radiotherapy (IMRT) in which radiation may be delivered in a helical fashion. However, unexpected pneumonitis may occur in patients treated with tomotherapy, especially in combination with chemotherapy, as a result of extensive low-dose radiation of large lung volumes. The aim of our study was to investigate the risk of radiation-induced pneumonitis after helical-mode and static-mode tomotherapy in patients with lung cancer and in an animal model. METHOD: A total of 63 patients with primary lung cancer who were treated with static or helical tomotherapy with or without concurrent chemoradiotherapy (CCRT) were analyzed. Additionally, rats with radiation-induced pulmonary toxicity, which was induced by the application of helical or static tomography with or without CCRT, were evaluated. RESULTS: Helical-mode tomotherapy resulted in a significantly higher rate of late radiation pneumonitis in lung cancer patients than static-mode tomotherapy when evaluated by the Radiation Therapy Oncology Group (RTOG) and National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) scoring system. In the animal model, helical tomotherapy alone induced significantly higher expression of interleukin (IL)-1α, IL-1ß, IL-6, and transforming growth factor (TGF)-ß in lung specimens, especially on the untreated side, compared to static tomotherapy alone. Additionally, rats treated with helical tomotherapy and CCRT demonstrated significantly higher expression of inflammatory cytokines compared to those treated with static tomotherapy and CCRT. CONCLUSION: Rat models treated with tomotherapy with or without CCRT could present similar patterns of pulmonary toxicity to those shown in lung cancer patients. The models can be used in further investigations of radiation induced pulmonary toxicity.
Subject(s)
Lung Neoplasms/radiotherapy , Radiation Pneumonitis/etiology , Radiotherapy, Intensity-Modulated/adverse effects , Adult , Aged , Aged, 80 and over , Animals , Chemoradiotherapy/adverse effects , Cytokines/biosynthesis , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immunohistochemistry , Inflammation , Male , Middle Aged , Radiation Pneumonitis/immunology , Radiotherapy, Intensity-Modulated/methods , Rats , Rats, Sprague-DawleyABSTRACT
As an angiogenic factor, thymidine phosphorylase (TP) expression in primary tumours has been thought to be a risk factor for lymph node (LN) and hepatic metastasis in patients with gastric adenocarcinoma. However, the molecular basis for the induction of metastasis by TP is largely unknown. We aim to elucidate the role of TP expression in gastric cancer neovascularisation and LN metastasis.The angiogenic and lymphangiogenic activity (CD31, D2-40, Ki-67, VEGFC, VEGFR3) and expression status of TP were detected in 103 resected human gastric carcinoma samples by immunohistochemistry. The influence of TP expression on neovascularisation and cancer cell invasion was further comparatively investigated in two groups of nude mice intraperitoneally injected with TP overexpressing MKN-45 cells (MKN-45/TP) and control cells (MKN-45/CV). In gastric cancer tissues, we found that high TP expression and various angiogenic and lymphangiogenic activities were significantly associated with poor prognostic outcomes. In addition, TP expression was also found to be associated with neovascularisation activity of gastric cancer tissues. In vivo, the MKN-45/TP group exhibited significantly increased infiltrating tumour nodules and neovascularisation activity compared to the MKN-45/CV group. TP could strongly influence gastric cancer progression via the dual activities of angiogenesis and lymphangiogenesis.
Subject(s)
Adenocarcinoma/pathology , Lymphangiogenesis/physiology , Neovascularization, Pathologic/enzymology , Stomach Neoplasms/pathology , Thymidine Phosphorylase/metabolism , Adenocarcinoma/enzymology , Adenocarcinoma/mortality , Adult , Aged , Animals , Blotting, Western , Female , Heterografts , Humans , Immunohistochemistry , Kaplan-Meier Estimate , Male , Mice , Mice, Nude , Middle Aged , Prognosis , Stomach Neoplasms/enzymology , Stomach Neoplasms/mortalityABSTRACT
Advanced gastric cancer is one of the most aggressive gastrointestinal malignancies, and ADAM (A disintegrin and metalloproteinase)-9 is a cell-surface membrane glycoprotein with oncogenic properties that is overexpressed in several cancers. Herein, we investigated the biologic mechanism of ADAM9 in the progression, proliferation, and invasion of gastric cancer. First, we detected ADAM's expression, processing, and protease activity in gastric cancer cells. Protease activity was moderately correlated with ADAM9 protein expression, but was better related to a processed smaller molecular weight (84 kDa) form of ADAM9. Knockdown of ADAM9 or specifically targeted monoclonal antibody (RAV-18) suppressed cancer cell proliferation and invasion in high ADAM9-expressing cells, not in low ADAM9-expressing cells. RAV-18 showed in vivo antitumor activity in a gastric cancer xenograft model. Hypoxia (1% oxygen) induced ADAM9 expression and functional activity in low ADAM9-expressing gastric cancer cells that was inhibited by siRNA knockdown or RAV-18 antibody to levels in normoxic cells. Overall, our studies show that ADAM9 plays an important role in gastric cancer proliferation and invasion, and that while expressed in some gastric cancer cells at high levels that are responsive to functional inhibition and antitumor activity of a catalytic site-directed antibody, other gastric cancer cells have low levels of expression and only when exposed to hypoxia do ADAM9 levels increase and the cells become responsive to ADAM9 antibody inhibition. Therefore, our findings suggest that ADAM9 could be an effective therapeutic target for advanced gastric cancer.
Subject(s)
ADAM Proteins/metabolism , Disintegrins/metabolism , Membrane Proteins/metabolism , Stomach Neoplasms/metabolism , Stomach Neoplasms/pathology , ADAM Proteins/antagonists & inhibitors , ADAM Proteins/genetics , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Movement , Cell Proliferation , Disease Models, Animal , Disease Progression , Enzyme Activation , Female , Gene Expression , Gene Expression Profiling , Heterografts , Humans , Hypoxia/metabolism , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Mice , Neoplasm Invasiveness , Protein Binding , RNA Interference , Stomach Neoplasms/drug therapy , Stomach Neoplasms/genetics , Tumor Burden/drug effectsABSTRACT
To investigate genes regulated by COX-2 or a COX-2 specific inhibitor, celecoxib, in irradiated cancer cells, we analyzed changes in gene expression using complementary DNA microarray following celecoxib or combined celecoxib and ionizing radiation (IR) treatment in a stable COX-2 knockdown A549 (AS) and a mock cell line (AN). Thirty-six genes were differentially expressed by COX-2 knockdown. Celecoxib changed the expressions of 40 and 69 genes in AN and AS cells, respectively. Twenty-seven genes were synchronously regulated by COX-2 and celecoxib. Among these, celecoxib regulated ras homolog gene family B and mitosin protein expression in a COX-2 dependent manner, especially in irradiated cells. In addition, we identified 11 genes that changed by more than 1.5 times the expected additive values after celecoxib and IR treatment. The current study may provide evidence that COX-2 or celecoxib regulates various intracellular functions in addition to their enzymatic activity regulation. We also identified candidate molecules that may be responsible for COX-2-dependent radiosensitization by celecoxib.