High extracellular glucose promotes cell motility by modulating cell deformability and contractility via the cAMP-RhoA-ROCK axis in human breast cancer cells.

The mechanical properties, or mechanotypes, of cells are largely determined by their deformability and contractility. The ability of cancer cells to deform and generate contractile force is critical in multiple steps of metastasis. Identifying soluble cues that regulate cancer cell mechanotypes and understanding the underlying molecular mechanisms regulating these cellular mechanotypes could provide novel therapeutic targets to prevent metastasis. Although a strong correlation between high glucose level and cancer metastasis has been demonstrated, the causality has not been elucidated, and the underlying molecular mechanisms remain largely unknown. In this study, using novel high-throughput mechanotyping assays, we show that human breast cancer cells become less deformable and more contractile with increased extracellular glucose levels (>5 mM). These altered cell mechanotypes are due to increased F-actin rearrangement and nonmuscle myosin II (NMII) activity. We identify the cAMP-RhoA-ROCK-NMII axis as playing a major role in regulating cell mechanotypes at high extracellular glucose levels, whereas calcium and myosin light-chain kinase (MLCK) are not required. The altered mechanotypes are also associated with increased cell migration and invasion. Our study identifies key components in breast cancer cells that convert high extracellular glucose levels into changes in cellular mechanotype and behavior relevant in cancer metastasis.

Defect in cytosolic Neu2 sialidase abrogates lipid metabolism and impairs muscle function in vivo.

Sialic acid (SA) is present in glycoconjugates and important in cell-cell recognition, cell adhesion, and cell growth and as a receptor. Among the four mammalian sialidases, cytosolic NEU2 has a pivotal role in muscle and neuronal differentiation in vitro. However, its biological functions in vivo remain unclear due to its very low expression in humans. However, the presence of cytoplasmic glycoproteins, gangliosides, and lectins involved in cellular metabolism and glycan recognition has suggested the functional importance of cytosolic Neu2 sialidases. We generated a Neu2 knockout mouse model via CRISPR/Cas9-mediated genome engineering and analyzed the offspring littermates at different ages to investigate the in vivo function of cytosolic Neu2 sialidase. Surprisingly, knocking out the Neu2 gene in vivo abrogated overall lipid metabolism, impairing motor function and leading to diabetes. Consistent with these results, Neu2 knockout led to alterations in sialylated glycoproteins involved in lipid metabolism and muscle function, as shown by glycoproteomics analysis.

Stem Cell Factor SOX9 Interacts with a Cell Death Regulator RIPK1 and Results in Escape of Cancer Stem Cell Death.

High-grade ovarian cancer (HGOC) is the most lethal gynecological cancer, with high metastasis and recurrence. Cancer stem cells (CSCs) are responsible for its apoptosis resistance, cancer metastasis, and recurrence. Thus, targeting CSCs would be a promising strategy for overcoming chemotherapy resistance and improving patient prognosis in HGOC. Among upregulated oncogenic proteins in HGOC, we found that transcription factor SOX9 showed a strong correlation with stemness-regulating ALDH1A1 and was localized predominantly in the cytoplasm of HGOC with lymph node metastasis. In order to address the role of unusual cytoplasmic SOX9 and to explore its underlying mechanism in HGOC malignancy, a Y2H assay was used to identify a necroptotic cell death-associated cytoplasmic protein, receptor-interacting serine/threonine protein kinase 1 (RIPK1), as a novel SOX9-interacting partner and further mapped their respective interacting domains. The C-terminal region containing the transactivation domain of SOX9 interacted with the death domain of R1PK1. Consistent with its stemness-promoting function, SOX9 knockdown in vitro resulted in changes in cell morphology, cell cycle, stem cell marker expression, cell invasion, and sphere formation. Furthermore, in vivo knockdown completely inhibited tumor growth in mouse xenograft model. We propose that cytoplasmic SOX9-mediated cell death suppression would contribute to cancer stem cell survival in HGOC.

Is Methionyl-tRNA Synthetase Applicable as a Diagnostic Marker for Lung Cancer in Bronchial Ultrasound-Guided Brushing Cells?

BACKGROUND AND OBJECTIVE: Methionyl-tRNA synthetase (MARS) and A variant of Aminoacyl-tRNA synthetase interacting multifunctional protein 2 (AIMP2) with an exon 2 deletion (AIMP2-DX2) are known to be overexpressed in lung cancer. However, their role as diagnostic markers in lung cancer has not been well established. Thus, we evaluated their diagnostic performance in brushed cells obtained from nodular lung lesions suspected of lung cancer. METHODS: Samples obtained by radial endobronchial ultrasound-guided brushing were processed for cytological examination with Papanicolaou (Pap) staining. Then, double IF staining with MARS and AIMP2-DX2 antibodies was measured in the cytology samples for peripheral lung nodules. The diagnostic performance was compared against biomarkers. RESULTS: MARS IF staining was the only independent staining method used for the prediction of malignant cells. The area under the curve (AUC) of conventional cytology, MARS IF, and MARS IF plus cytology was 0.64, 0.68, and 0.69, respectively. The diagnostic accuracy was increased in MARS IF plus conventional cytology compared with cytology alone (71% vs. 47%). CONCLUSIONS: The combination of MARS staining with conventional cytology showed increases in the diagnostic accuracy for diagnosing lung nodules suspected of lung cancer on chest-computed tomography scans.

Cardiophrenic Lymph Node Metastasis of Ovarian High-grade Serous Carcinoma Showing Wild-type p53 Immunostaining Pattern and Aberrant CD56 Expression.

Patterns of p53 immunostaining are used as a surrogate marker for tumor protein 53 (TP53) mutations in the diagnosis of ovarian high-grade serous carcinoma (HGSC). We present a rare case of ovarian HGSC that metastasized to the diaphragm and cardiophrenic lymph nodes and showed the immunostaining pattern of wild-type p53 and aberrant neural cell adhesion molecule (CD56) expression. A 63-year-old woman developed multifocal metastases in the diaphragmatic pleura and cardiophrenic lymph nodes. Because she had a history of ovarian HGSC and pulmonary adenocarcinoma, we considered the possibility that the metastatic carcinoma was of either ovarian or pulmonary origin. Immunostaining revealed that the tumor cells were negative for thyroid transcription factor 1 but positive for Wilms tumor 1. The tumor additionally exhibited strong membranous CD56 expression and patchy p53 expression, both of which were inconsistent with the characteristics of ovarian HGSC. However, targeted sequencing analysis revealed that the tumor harbored a pathogenic mutation at the splice acceptor site of TP53 intron 9 (c.994-1G>C).

Homeoprotein Msx1-PIASy Interaction Inhibits Angiogenesis.

Previously, we demonstrated that the homeoprotein Msx1 interaction with p53 inhibited tumor growth by inducing apoptosis. However, Msx1 can exert its tumor suppressive effect through the inhibition of angiogenesis since growth of the tumor relies on sufficient blood supply from the existing vessels to provide oxygen and nutrients for tumor growth. We hypothesized that the inhibition of tumor growth by Msx1 might be due to the inhibition of angiogenesis. Here, we explored the role of Msx1 in angiogenesis. Overexpression of Msx1 in HUVECs inhibited angiogenesis, and silencing of Msx1 by siRNA abrogated its anti-angiogenic effects. Furthermore, forced expression of Msx1 in mouse muscle tissue inhibited vessel sprouting, and application of an Ad-Msx1-transfected conditioned medium onto the chicken chorioallantoic membrane (CAM) led to a significant inhibition of new vessel formation. To explore the underlying mechanism of Msx1-mediated angiogenesis, yeast two-hybrid screening was performed, and we identified PIASy (protein inhibitor of activated STAT Y) as a novel Msx1-interacting protein. We mapped the homeodomain of Msx1 and the C-terminal domain of PIASy as respective interacting domains. Consistent with its anti-angiogenic function, overexpression of Msx1 suppressed the reporter activity of VEGF. Interestingly, PIASy stabilized Msx1 protein, whereas deletion of the Msx1-interacting domain in PIASy abrogated the inhibition of tube formation and the stabilization of Msx1 protein. Our findings suggest the functional importance of PIASy-Msx1 interaction in Msx1-mediated angiogenesis inhibition.

Non-proteolytic calpain-6 interacts with VEGFA and promotes angiogenesis by increasing VEGF secretion.

Angiogenesis is involved in both normal physiological and pathological conditions. Vascular endothelial growth factor (VEGF) is a major factor for promoting angiogenesis. The current anti-VEGF therapies have limited efficacy and significant adverse effects. To find novel targets of VEGFA for angiogenesis inhibition, we performed yeast two-hybrid screening and identified calpain-6 as a novel VEGFA-interaction partner and confirmed the endogenous VEGFA-calpain-6 interaction in mammalian placenta. A domain mapping study revealed that the Gly321-Asp500 domain in calpain-6 is required for the interaction with the C-terminus of the VEGFA protein. The functional significance of the VEGFA-calpain-6 interaction was explored by assessing its effect on angiogenesis in vitro. Whereas forced overexpression of calpain-6 increased the secretion of the VEGF protein and tube formation, knockdown of calpain-6 expression abrogated the calpain-6-mediated VEGF secretion and tube formation in HUVECs. Consistent with the domain mapping result, overexpressing calpain-6 without the VEGFA-interacting domain III (Gly321-Asp500) failed to increase the secretion of VEGF protein. Our results identify calpain-6, an unconventional non-proteolytic calpain, as a novel VEGFA-interacting protein and demonstrate that their interaction is necessary to enhance VEGF secretion. Thus, calpain-6 might be a potential molecular target for angiogenesis inhibition in many diseases.

A Case of Pulmonary Cryptococcosis in an Immunocompetent Male Patient Diagnosed by a Percutaneous Supraclavicular Lymph Node Biopsy.

Cryptococcal pneumonia usually occurs in immunocompromised patients with malignancy, acquired immune deficiency syndrome, organ transplantations, immunosuppressive chemotherapies, catheter insertion, or dialysis. It can be diagnosed by gaining tissues in lung parenchyma or detecting antigen in blood or bronchoalveolar lavage fluid. Here we report an immunocompetent 32-year-old male patient with diabetes mellitus diagnosed with cryptococcal pneumonia after a ultrasound-guided percutaneous supraclavicular lymph node core needle biopsy. We treated him with fluconazole at 400 mg/day for 9 months according to the guideline. This is the first case that cryptococcal pneumonia was diagnosed from a percutaneous lymph node biopsy in South Korea.

Potential masking of airway eosinophilic inflammation by combination therapy in asthma.

PURPOSE: Long-acting ß2 agonists (LABA) may mask ongoing bronchial inflammation, leaving asthmatic patients at greater risk of severe complications. The aim of this study was to compare the effect of combination therapy using low-dose inhaled corticosteroids (ICS) plus LABA on airway inflammation in asthma to the effect of medium-dose ICS alone. METHODS: Twenty-four patients with asthma not controlled by low-dose (400 µg per day) budesonide alone were enrolled in this prospective crossover study. Patients were randomized into 2 treatment phases: one receiving medium-dose (800 µg per day) budesonide (ICS phase), and the other receiving a combination therapy of low-dose budesonide/formoterol (360 µg/9 µg per day) delivered by a single inhaler (LABA phase). Each treatment phase lasted for 6 week, after which patients were crossed over. Asthma symptoms, lung function, and airway inflammation were compared between the 2 phases. RESULTS: Twenty-three patients completed the study; adequate sputum samples were collected from 17 patients. Asthma symptoms and lung function remained similar between the 2 phases. However, the mean sputum eosinophil percentage was higher in the LABA phase than in the ICS phase (5.07±3.82% vs. 1.02±1.70%; P<0.01). Sputum eosinophilia (≥3%) was more frequently observed in the LABA phase than in the ICS phase (six vs. two). CONCLUSION: Addition of LABA may mask airway eosinophilic inflammation in asthmatic patients whose symptoms are not controlled with low-dose ICS.

Comparison of the causes and clinical features of drug rash with eosinophilia and systemic symptoms and stevens-johnson syndrome.

PURPOSE: Drug rash with eosinophilia and systemic symptoms (DRESS) and the Stevens-Johnson syndrome (SJS) are both severe drug reactions. Their pathogenesis and clinical features differ. This study compared the causes and clinical features of SJS and DRESS. METHODS: We enrolled 31 patients who were diagnosed with DRESS (number=11) and SJS (number=20). We retrospectively compared the clinical and laboratory data of patients with the two disorders. RESULTS: In both syndromes, the most common prodromal symptoms were itching, fever, and malaise. The liver was commonly involved in DRESS. The mucosal membrane of the oral cavity and eyes was often affected in SJS. The most common causative agents in both diseases were antibiotics (DRESS 4/11 (37%), SJS 8/20 (40%)), followed by anticonvulsants (DRESS 3/11 (27%), SJS 7/20 (35%)). In addition, dapsone, allopurinol, clopidogrel, sulfasalazine and non-steroidal anti-inflammatory drugs (NSAIDs) were sporadic causes. CONCLUSIONS: The most common causes of DRESS and SJS were antibiotics, followed by anticonvulsants, NSAIDs and sulfonamides. The increase in the use of antibiotics in Korea might explain this finding.

Toxocariasis might be an important cause of atopic myelitis in Korea.

Atopic myelitis is defined as myelitis with atopic diasthesis but the cause is still unknown. Toxocariasis is one of the common causes of hyperIgEaemia that may lead to neurologic manifestations. The purpose of this study was to evaluate the sero-prevalence of Toxocara specific IgG Ab among the atopic myelitis patients. We evaluated the medical records of 37 patients with atopic myelitis whose conditions were diagnosed between March 2001 and August 2007. Among them, the 33 sera were analyzed for specific serum IgG Ab to Toxocara excretory-secretory antigens (TES). All of 37 patients had hyperIgEaemia. Specific IgE to D. pteronyssinus and D. farinae was detected in 22 (64.7%) and 34 (100%) patients, respectively, of the 34 patients. Thirty-one of 33 patients (93.9%) were found to be positive by TES IgG enzyme-linked immunosorbent assay (ELISA). Based on the image findings of eosinophilic infiltrations in the lung and liver, 8 patients had positive results. These results inferred that the prevalence of toxocariasis was high in patients with atopic myelitis. Our results suggest that toxocariasis might be an important cause of atopic myelitis and Toxocara ELISA is essential for evaluating the causes of atopic myelitis.

Exhaled nitric oxide measurement is useful for the exclusion of nonasthmatic eosinophilic bronchitis in patients with chronic cough.

BACKGROUND: Nonasthmatic eosinophilic bronchitis (NAEB) is an important cause of chronic cough, and it can be diagnosed by an induced-sputum (IS) examination. However, an IS examination is a complex and time-consuming procedure, and it has limited clinical application. This study aimed to evaluate the role of exhaled nitric oxide (NO) for the investigation of chronic cough, especially of NAEB. METHODS: Two hundred eleven nonsmoking patients with a cough lasting > 3 weeks were enrolled in the study. The patients were examined and investigated with conventional diagnostic tools, including an IS examination. Exhaled NO was measured by a chemoluminescent analyzer. RESULTS: One hundred seventeen patients with adequate IS results were analyzed: asthma, n = 14; NAEB, n = 21; and "others," n = 82. Exhaled NO and IS eosinophils were significantly higher in the asthma group and NAEB group than in the others group. Exhaled NO and IS eosinophils were significantly correlated in the asthma and NAEB groups. In the nonasthmatic group, the sensitivity and specificity of exhaled NO for detecting NAEB, using 31.7 parts per billion as the exhaled NO cutoff point, were 86% and 76%, respectively. Positive and negative predictive values were 47% and 95%, respectively, and positive and negative likelihood ratios were 3.51 and 0.19, respectively. CONCLUSION: We concluded that exhaled NO measurement may be useful as part of the initial evaluation for chronic cough, especially for the exclusion of NAEB. A low level of exhaled NO suggested little likelihood of NAEB for the nonasthmatic patients with chronic cough.

DNA methylation and the expression of IL-4 and IFN-gamma promoter genes in patients with bronchial asthma.

Although CpG methylation is thought to be a negative regulator of gene transcription, its relationship with cytokine expression remains unclear. Interleukin (IL)-4 and interferon (IFN)-gamma are major cytokines that affect the differentiation of naïve CD4+ T lymphocytes into the Th1 and Th2 lineage. We used bisulfite deoxyribonucleic acid modification and sequencing to examine the relationship between CpG methylation and IL-4 and IFN-gamma gene expression before and after allergen stimulation in human CD4+ T lymphocytes from sensitized hosts. In naïve cells, the CpGs in the promoter regions were methylated largely in both the IL-4 and IFN-gamma genes. After Dermatophagoides pteronyssinus/Dermatophagoides farinae stimulation, the degree of unmethylation in the IL-4 gene increased in cells from patients with bronchial asthma. After phytohemagglutinin stimulation, the degree of unmethylation increased in cells from nonallergic control subjects. The concentration of IL-4 was strongly correlated with the degree of unmethylation in the patient group. These data suggest that CpGs located at -80 of the IL-4 gene and at -295, -186, and +122 of the IFN-gamma gene have regulatory activities important for cytokine expression. We conclude that the stimulation of CD4+ T lymphocytes causes considerable increases in the degree of unmethylation and that in sensitized hosts, the extent of unmethylation correlates with the concentration of IL-4.

Prevalence of lower airway diseases in patients with chronic rhinosinusitis.

CONCLUSION: There is high prevalence of lower airway diseases in patients with chronic rhinosinusitis and frequently co-existing lower airway diseases have not been diagnosed before. OBJECTIVES: To examine the prevalence of lower airway diseases in patients with chronic rhinosinusitis. METHODS: Seventy-three consecutive patients with chronic rhinosinusitis were enrolled in this prospective study. With routine physical examination, spirometry and methacholine bronchial provocation test were performed and chest simple radiograph or chest computed tomography was taken. RESULTS: Thirty patients (41.1%) had lower airway diseases. There were 8 patients with asthma, 5 with asymptomatic bronchial hyperresponsiveness, 11 with small airway disease, 2 with chronic obstructive pulmonary disease and 4 with bronchiectasis. Of these 30 patients, 21 patients (70.0%) were first diagnosed as having lower airway diseases in this study.

Causes and clinical features of subacute cough.

STUDY OBJECTIVES: Cough lasting > 3 weeks has been defined as chronic cough. However, it might be more persuasive to divide cough into subacute, lasting 3 to 8 weeks, and chronic, lasting > 8 weeks. We evaluated the causes and clinical courses of subacute cough, and the value of the bronchoprovocation test and induced sputum examination (IS). METHODS: Nonsmoking patients with cough of 3 to 8 weeks duration were enrolled into the study. Patients with dyspnea, basal FEV1 of < 70% predicted, abnormal findings on a plain chest radiograph or physical examination were excluded. We prescribed an antihistamine-decongestant for patients who were suspected to have postinfectious cough or postnasal drip. If patients had positive results on a bronchoprovocation test or IS, therapy with inhaled corticosteroids was substituted according to an algorithmic approach. RESULTS: One hundred eighty-four patients (77 men and 107 women) were evaluated; the mean age of the study group was 47.5 years. Eighty-nine of 184 patients had postinfectious cough. Cough resolved without treatment in 62 patients. Twenty-nine of 43 patients with positive bronchoprovocation test results had cough-variant asthma. CONCLUSIONS: Postinfectious cough was the most common cause of subacute cough. The spontaneous resolution of cough was frequent in patients with subacute cough. Unless asthma was strongly suspected, the performance of the bronchoprovocation test could be delayed until after empirical treatment had been administered.

The prevalence and diagnostic value of toxocariasis in unknown eosinophilia.

Toxocariasis is one of the causes of eosinophilia in peripheral blood and provokes eosinophilic infiltration in internal organs. Extended studies on the prevalence and clinical characteristics of toxocariasis have been rare. The aim of this study is to evaluate the prevalence of toxocariasis in unknown eosinophilia and to analyze the efficacy of toxocara enzyme-linked immunosorbent assay (ELISA). We evaluated patients presenting with peripheral blood eosinophilia (>500 cells/microl or > or =10% of white blood cell count). After checking drug histories and the presence of allergic diseases and parasitic infections, specific serum IgG antibody to Toxocara canis larval antigen was measured by ELISA. Liver and lung involvement was also evaluated. One-hundred and three patients were evaluated, and the mean age was 50.9 years old. Seventy patients (68.0%) were diagnosed as having toxocariasis. The patients who had a history of raw liver eating had a higher incidence, and the patients with liver involvement had higher serum eosinophil cationic protein values. The eosinophil count was normalized in 7 of 16 treated patients and in 25 of 54 untreated patients. The mean improvement duration was 12 months. We concluded that the prevalence of toxocariasis was high in patients with unknown eosinophilia, and the toxocara ELISA was essential for evaluation of the causes of unknown eosinophilia.