House dust mite-induced Akt-ERK1/2-C/EBP beta pathway triggers CCL20-mediated inflammation and epithelial-mesenchymal transition for airway remodeling

House dust mite (HDM) allergens cause inflammatory responses and chronic allergic diseases such as bronchial asthma and atopic dermatitis. Here, we investigate the mechanism by which HDM induces C-C chemokine ligand 20 (CCL20) expression to promote chronic inflammation and airway remodeling in an HDM-induced bronchial asthma mouse model. We showed that HDM increased CCL20 levels via the Akt-ERK1/2-C/EBPβ pathway. To investigate the role of CCL20 in chronic airway inflammation and remodeling, we made a mouse model of CCL20-induced bronchial asthma. Treatment of anti-CCL20Ab in this mouse model showed the reduced airway hyper-responsiveness and inflammatory cell infiltration into peribronchial region by neutralizing CCL20. In addition, CCL20 induced the Nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation through NLRP3 deubiquitination and transcriptional upregulation in BEAS-2B cells. As expected, anti-CCL20Ab markedly suppressed NLRP3 activation induced by CCL20. Moreover, HDM-induced CCL20 leads to epithelial-mesenchymal transition in the lung epithelium which appears to be an important regulator of airway remodeling in allergic asthma. We also found that anti-CCL20Ab attenuates airway inflammation and remodeling in an HDM-induced mouse model of bronchial asthma. Taken together, our results suggest that HDM-induced CCL20 is required for chronic inflammation that contributes airway remodeling in a mouse model of asthma.ope

Comparison of liver oncogenic potential among human RAS isoforms.

Mutation in one of three RAS genes (i.e., HRAS, KRAS, and NRAS) leading to constitutive activation of RAS signaling pathways is considered a key oncogenic event in human carcinogenesis. Whether activated RAS isoforms possess different oncogenic potentials remains an unresolved question. Here, we compared oncogenic properties among RAS isoforms using liver-specific transgenesis in mice. Hydrodynamic transfection was performed using transposons expressing short hairpin RNA downregulating p53 and an activated RAS isoform, and livers were harvested at 23 days after gene delivery. No differences were found in the hepatocarcinogenic potential among RAS isoforms, as determined by both gross examination of livers and liver weight per body weight ratio (LW/BW) of mice expressing HRASQ61L, KRAS4BG12V and NRASQ61K. However, the tumorigenic potential differed significantly between KRAS splicing variants. The LW/BW ratio in KRAS4AG12V mice was significantly lower than in KRAS4BG12V mice (p < 0.001), and KRAS4AG12V mice lived significantly longer than KRRAS4BG12V mice (p < 0.0001). Notably, tumors from KRAS4AG12V mice displayed higher expression of the p16INK4A tumor suppressor when compared with KRAS4BG12V tumors. Forced overexpression of p16INK4A significantly reduced tumor growth in KRAS4BG12V mice, suggesting that upregulation of p16INK4A by KRAS4AG12V presumably delays tumor development driven by the latter oncogene.ope

Overexpression of CEACAM6 activates Src-FAK signaling and inhibits anoikis, through homophilic interactions in lung adenocarcinomas

Among carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family proteins, CEACAM6 has received less attention than CEACAM5 and its presence and role in lung cancer are largely unknown. The application of CellphoneDB on the single cell RNA sequencing dataset showed that the homophilic interactions among CEACAM6 molecules, which are overexpressed in lung cancer cells were highly significant. CEACAM6 was overexpressed in 80.1% of lung adenocarcinomas and its overexpression had a significant relationship with non-smoking history and activating EGFR mutations. The effect of CEACAM6 overexpression on patient prognosis was evaluated using TCGA-LUAD dataset; the CEACAM6 overexpression group showed a shorter overall survival than that of the control group when matched for stage, age, sex, and pack-years. Immunoblotting of cell culture soup and ELISA of human derived material suggested that the majority of CEACAM6 was present on the cancer cell surface and interacted with other cancer cells in the crowded tumor microenvironment. Treatment with CEACAM6 showed CEACAM6 homophilic interactions in the cell membrane and anoikis inhibition through the activation of the Src-FAK pathway. Inhibition of CEACAM6 or its homophilic interactions in the cancer cell membrane may provide another therapeutic strategy for lung cancer.ope

Development of a transgenic mouse model of hepatocellular carcinoma with a liver fibrosis background

BACKGROUND: Liver fibrosis and its end-stage disease, cirrhosis, are major risk factors for hepatocellular carcinoma (HCC) and present in 80 to 90 % of patients with HCC. Current genetically engineered mouse models for HCC, however, generally do not feature liver fibrosis, which is a critical discrepancy between human HCC and murine models thereof. In this study, we developed a simple transgenic mouse model of HCC within the context of a fibrotic liver. METHODS: Employing hydrodynamic transfection (HT), coupled with the Sleeping Beauty (SB) transposon system, liver was stably transfected with transposons expressing cMyc and a short hairpin RNA down-regulating p53 (shp53). A chronic liver injury model, induced by hepatotoxic carbon tetrachloride (CCl4), was applied to the transgenic mice, allowing cells expressing cMyc plus shp53 to become malignant in the background of liver fibrosis. RESULTS: Livers harvested about 3 months after HT had excessive collagen deposition and activated hepatic stellate cells surrounding the tumors. Hepatocarcinogenesis was significantly accelerated in the fibrotic livers compared to those of the control, significantly decreasing the life span of the mice. The tumor incidence and average number of tumors per mouse were significantly higher in the group treated with CCl4 compared to the vehicle-treated control mice, following HT (p < 0.01). CONCLUSIONS: Considering the simplicity and efficiency in generating HCC for fibrotic livers, the transgenic HCC model has the potential to be effectively used in preclinical testing of HCC anticancer therapy and in studies of hepatocarcinogenesis in fibrotic livers.ope

Transgenic mouse model expressing P53(R172H), luciferase, EGFP, and KRAS(G12D) in a single open reading frame for live imaging of tumor

Genetically engineered mouse cancer models allow tumors to be imaged in vivo via co-expression of a reporter gene with a tumor-initiating gene. However, differential transcriptional and translational regulation between the tumor-initiating gene and the reporter gene can result in inconsistency between the actual tumor size and the size indicated by the imaging assay. To overcome this limitation, we developed a transgenic mouse in which two oncogenes, encoding P53(R172H) and KRAS(G12D), are expressed together with two reporter genes, encoding enhanced green fluorescent protein (EGFP) and firefly luciferase, in a single open reading frame following Cre-mediated DNA excision. Systemic administration of adenovirus encoding Cre to these mice induced specific transgene expression in the liver. Repeated bioluminescence imaging of the mice revealed a continuous increase in the bioluminescent signal over time. A strong correlation was found between the bioluminescent signal and actual tumor size. Interestingly, all liver tumors induced by P53(R172H) and KRAS(G12D) in the model were hepatocellular adenomas. The mouse model was also used to trace cell proliferation in the epidermis via live fluorescence imaging. We anticipate that the transgenic mouse model will be useful for imaging tumor development in vivo and for investigating the oncogenic collaboration between P53(R172H) and KRAS(G12D).ope

Early lung carcinogenesis and tumor microenvironment observed by single-cell transcriptome analysis

With the increasing interest in health screening with chest CT Ground-glass nodule (GGN) has become one of the common lung lesions encountered in daily medical practice. Because lung adenocarcinoma in the form of GGN is an ideal model for studying early lung carcinogenesis, 11 GGN and normal lung specimens from 6 never smoker patients were studied by single-cell RNA sequencing. Lung cancer cells showed enrichment of gene sets related to small vesicle processing and surfactant homeostasis compared to non-malignant lung epithelial cells, suggesting the dysregulation of surfactant pathway may be involved in early lung carcinogenesis. Along with cancer-associated fibroblasts showing enrichment of gene sets involved in negative regulation of protein kinase activity and negative regulation of endothelial cell proliferation, tumor microenvironment (TME) was dominated by infiltration of TNFRSF4+/TNFRSF18+/CTLA4+ regulatory T cells (Treg) and depletion of CD8+ cytotoxic T cells (TC) and γδTC. Majority of mucosa-associated lymphoid tissue B cells (BCs) and follicular BCs were detected within tumor tissue, which was associated with CXCL13 overexpressed in intratumoral Tregs and CD4+ memory TCs. Coordination of components of the TME towards immune evasion is governed by Tregs from the onset of lung cancer, requiring unremitting efforts to target and overcome them. This provision of information on changes in cancer cell-specific biomarkers and TME using early lung cancer from never smokers will provide new insight into early lung carcinogenesis and useful targets for treatment.ope

Investigation of Oncogenic Cooperation in Simple Liver-Specific Transgenic Mouse Models Using Noninvasive In Vivo Imaging

iver cancer is a complex multistep process requiring genetic alterations in multiple proto-oncogenes and tumor suppressor genes. Although hundreds of genes are known to play roles in hepatocarcinogenesis, oncogenic collaboration among these genes is still largely unknown. Here, we report a simple methodology by which oncogenic cooperation between cancer-related genes can be efficiently investigated in the liver. We developed various non-germline transgenic mouse models using hydrodynamics-based transfection which express HrasG12V, SmoM2, and a short-hairpin RNA down-regulating p53 (shp53) individually or in combination in the liver. In this transgenic system, firefly luciferase was co-expressed with the oncogenes as a reporter, allowing tumor growth in the liver to be monitored over time without an invasive procedure. Very strong bioluminescence imaging (BLI) signals were observed at 4 weeks post-hydrodynamic injection (PHI) in mice co-expressing HrasG12V and shp53, while only background signals were detected in other double or single transgenic groups until 30 weeks PHI. Consistent with the BLI data, tumors were observed in the HrasG12V plus shp53 group at 4 weeks PHI, while other transgenic groups failed to exhibit a hyperplastic nodule at 30 weeks PHI. In the HrasG12V plus shp53 transgenic group, BLI signals were well-correlated with actual tumor growth in the liver, confirming the versatility of BLI-based monitoring of tumor growth in this organ. The methodology described here is expected to accelerate and facilitate in vivo studies of the hepatocarcinogenic potential of cancer-related genes by means of oncogenic cooperation.ope

Antifibrotic effects of magnesium lithospermate B on hepatic stellate cells and thioacetamide-induced cirrhotic rats.

Magnesium lithospermate B (MLB) is one of the major active components of Salvia miltiorrhizae. The anti-oxidative effects of Salvia miltiorrhizae have been previously reported. The aim of this study was to investigate the effect of purified MLB on hepatic fibrosis in rats and on the fibrogenic responses in hepatic stellate cells (HSCs). Hepatic fibrosis was induced in rats by intraperitoneal thioacetamide (TAA) injections over a period of 8 or 12 weeks. MLB was orally administered daily by gavage tube. Serum AST and ALT levels in TAA+ MLB group were significantly lower than those in TAA only group at week 8. Hepatic fibrosis was significantly attenuated in TAA+MLB group than in TAA only group at week 8 or 12. Activation of HSCs was also decreased in TAA+MLB group as compared to TAA only group. Hepatic mRNA expression of α-smooth muscle actin (α-SMA), TGF-β1, and collagen α1(I) was significantly decreased in TAA+MLB group as compared to TAA only group. Incubation with HSCs and MLB (>or=100 μM) for up to 48 h showed no cytotoxicity. MLB suppressed PDGF-induced HSC proliferation. MLB inhibited NF-ΚB transcriptional activation and monocyte chemotactic protein 1 (MCP-1) production in HSCs. MLB strongly suppressed H(2)O(2)-induced reactive oxygen species (ROS) generation in HSCs, and MLB inhibited type I collagen secretion in HSCs. We concluded that MLB has potent antifibrotic effect in TAA-treated cirrhotic rats, and inhibits fibrogenic responses in HSCs. These data suggest that MLB has potential as a novel therapy for hepatic fibrosis.ope

Synergic chemoprevention with dietary carbohydrate restriction and supplementation of AMPK-activating phytochemicals: the role of SIRT1

Calorie restriction or a low-carbohydrate diet (LCD) can increase life span in normal cells while inhibiting carcinogenesis. Various phytochemicals also have calorie restriction-mimetic anticancer properties. We investigated whether an isocaloric carbohydrate-restriction diet and AMP-activated protein kinase (AMPK)-activating phytochemicals induce synergic tumor suppression. We used a mixture of AMPK-activating phytochemical extracts including curcumin, quercetin, catechins, and resveratrol. Survival analysis was carried out in a B16F10 melanoma model fed a control diet (62.14% kcal carbohydrate, 24.65% kcal protein and 13.2% kcal fat), a control diet with multiple phytochemicals (MP), LCD (16.5, 55.2, and 28.3% kcal, respectively), LCD with multiple phytochemicals (LCDmp), a moderate-carbohydrate diet (MCD, 31.9, 62.4, and 5.7% kcal, respectively), or MCD with phytochemicals (MCDmp). Compared with the control group, MP, LCD, or MCD intervention did not produce survival benefit, but LCDmp (22.80±1.58 vs. 28.00±1.64 days, P=0.040) and MCDmp (23.80±1.08 vs. 30.13±2.29 days, P=0.008) increased the median survival time significantly. Suppression of the IGF-1R/PI3K/Akt/mTOR signaling, activation of the AMPK/SIRT1/LKB1pathway, and NF-κB suppression were the critical tumor-suppression mechanisms. In addition, SIRT1 suppressed proliferation of the B16F10 and A375SM cells under a low-glucose condition. Alterations in histone methylation within Pten and FoxO3a were observed after the MCDmp intervention. In the transgenic liver cancer model developed by hydrodynamic transfection of the HrasG12V and shp53, MCDmp and LCDmp interventions induced significant cancer-prevention effects. Microarray analysis showed that PPARα increased with decreased IL-6 and NF-κB within the hepatocytes after an MCDmp intervention. In conclusion, an isocaloric carbohydrate-restriction diet and natural AMPK-activating agents induce synergistic anticancer effects. SIRT1 acts as a tumor suppressor under a low-glucose condition.ope

랫드 간경변 모델에서 방사선에 의한 간손상의 표적 단백질의 탐색

Dept. of Medical Science/석사[한글] 간세포암은 전세계적으로, 특히 아시아에서 흔하게 발생하는 암 중의 하나로써, 병의 빠른 진행과 간경변증의 동반과 같은 문제로 인하여 예후가 좋지 않다. 방사선 치료는 간세포암을 위한 여러 치료 방법들 중의 하나로, 초기 간경변증을 동반한 간세포암에서 방사선 치료가 시행되고 있다. 그러나 간경변증을 동반한 간에 방사선 조사로 인하여 간 기능이 악화되는 기전에 대한 체계적인 연구가 미흡한 상황이다.본 연구에서는 Thioacetamide (TAA)로 유도한 랫드 간경변 모델에 방사선을 조사하였을 때 간조직과 혈액에서 공통적으로 발현하는 단백질을 알아보고, 그 기능을 규명하고자 하였다.웅성 Wistar 랫드에 0.3 g/L TAA를 음용수에 희석하여 9주 동안 복용시켜 간경변을 유발하였고, 간경변을 가진 랫드의 일부는 10 Gy의 방사선을 간의 우엽에만 부분 조사하였다. 랫드는 방사선 조사 후 3주까지 관찰하였고, 획득한 간 조직과 혈액은 프로테오믹스 기법을 이용하여 분석하고 확인하였다.조직, 형태학적 분석을 통해 간경변증을 동반한 간에 방사선을 조사하면 간 섬유화가 증가됨을 볼 수 있었다. 간 조직과 혈액의 프로테오믹스 결과, 총 57개의 단백질이 방사선에 의해 발현의 차이를 보였다. 발현된 단백질은 기능별로 구별한 결과 면역반응, 신호변환, 세포사멸, 증식/분화, 대사에 관여하는 단백질로 나타났다. 방사선 조사 후, heparanase precursor는 증가하고 hepatocyte growth factor receptor는 감소하는 양상이 간 조직과 혈액에서 공통적으로 발현되었다. Proliferating cells nuclear antigen (PCNA)의 발현은 방사선 조사 후 감소하였으며, H&E 염색을 통해 관찰한 세포사멸은 방사선 조사 후에 증가하였다.TAA로 유도한 랫드 간경변 모델에서 방사선 조사로 인해 간섬유화와 세포사멸이 증가함을 관찰하였고, 간조직과 혈액 내 단백질의 변화를 확인하였다. 방사선 조사 후에 간 조직과 혈액에서 공통적으로 heparanase precursor가 증가하고 hepatocyte growth factor receptor가 감소되었으며, 이 단백질이 아마도 향후 방사선 치료에 의한 간 손상을 인지하고 치료를 개선할 수 있는 방법 개발에 유용할 것으로 생각된다. [영문]Hepatocellular carcinoma (HCC) is one of the most common malignancies in Asia as well as worldwide. It has a poor prognosis due to its rapid progression and the complicating liver cirrhosis. Radiation therapy (RT) is one of several treatments for HCC. It has been tried to HCC in mild-to-moderate degree of associated liver cirrhosis. However, information is insufficient on the tolerance of RT in liver cirrhosis. The aim of this study is to assess radiation effect on cirrhotic liver and to identify common proteins in liver tissue and serum following radiation exposure in rats with thioacetamide (TAA)-induced liver cirrhosis.Male Wistar rats were treated with 0.3 g/L TAA in their drinking water for 9 weeks. To determine if radiation was associated with hepatic injury, two experimental groups were examined: TAA-alone and TAA-plus-radiation, with three rats in each group. The livers of the cirrhotic rats were subjected to 10 Gy radiation and the animals were killed 3 weeks later. The development of liver cirrhosis was observed histologically. Proteins from liver tissue and serum were analyzed using two-dimensional electrophoresis and identified using quadrupole time of flight (Q-TOF) mass spectrometry (MS). Identified proteins were validated using Western blotting.Histological examination showed that hepatic fibrosis more increased following radiation in TAA-induced liver cirrhosis. In the proteomic analysis of liver tissue and serum, 57 proteins differed significantly between the TAA-alone and TAA-plus-radiation groups. The identified proteins had functions in the immune response, signal transduction, apoptosis, proliferation/differentiation,and reactive oxygen species metabolism. Common proteins in liver tissue and serum following radiation were the heparanase precursor and the hepatocyte growth factor receptor. Expression of the heparanase precursor increased twofold in the TAA-plus-radiation group compared to the TAA-alonegroup, while the expression of the hepatocyte growth factor receptor decreased twofold in the TAA-plus-radiation group. The expression of proliferating cell nuclear antigen (PCNA) decreased following radiation, just like the hepatocyte growth factor receptor. However, hematoxylin and eosin staining showed that apoptotic cells increased following radiation.In conclusion, radiation induced hepatic fibrosis and apoptosis in TAA-induced liver cirrhosis. The heparanase precursor increased and hepatocyte growth factor receptor decreased both in liver tissue and serum, suggesting that these proteins may be useful in detecting and monitoring radiation-induced hepatic injury.ope