Comparison of Disease Severity Classifications of Chronic Obstructive Pulmonary Disease: GOLD vs. STAR in Clinical Practice.

BACKGROUND: In chronic obstructive pulmonary disease (COPD), there are two known classifications for assessing what is called disease severity. One is the Global Initiative for Chronic Obstructive Lung Disease (GOLD) classification, which is based on the post-bronchodilator value of FEV1 (% reference). The other is the STaging of Airflow obstruction by Ratio (STAR), with four grades of severity in subjects with an FEV1/FVC ratio <0.70: STAR 1 ≥0.60 to <0.70, STAR 2 ≥0.50 to <0.60, STAR 3 ≥0.40 to <0.50, and STAR 4 <0.40. PURPOSE: The aim of this study was to compare the staging of COPD using the GOLD and STAR classifications in clinical practice. METHODS: We reanalyzed data from our outpatient cohort study, which included 141 participants with COPD from 2015 to 2023. We compared mortality and COPD-specific health status between the GOLD 1 to 4 groups and the STAR 1 to 4 groups. RESULTS: By simple calculation, GOLD and STAR severity classes coincided in 75 participants (53.2%). The weighted Bangdiwala B value with linear weights was 0.775. The participants were observed for up to 95 months, with a median of 54 months. Death was confirmed in 29 participants (20.5%). In univariate Cox proportional hazards analyses, there was a significant difference in mortality between the GOLD 1 and GOLD 3 + 4 groups, with the GOLD 1 group used as the reference [hazard ratio 4.222 (95% CI 1.298-13.733), p = 0.017]. However, there was no statistically significant predictive relationship between STAR 1 and STAR 2, or between STAR 1 and STAR 3 + 4. St. George's Respiratory Questionnaire (SGRQ) Total and COPD Assessment Test (CAT) scores were significantly different between all GOLD groups, except for the CAT score between GOLD 1 and GOLD 2. The SGRQ Total and CAT scores were significantly different between STAR 1 and STAR 3 + 4, but not between STAR 1 and STAR 2. CONCLUSION: From the perspective of all-cause mortality and COPD-specific health status, the GOLD classification is more discriminative than STAR.

Is Frailty a Mortality Predictor in Subjects with Chronic Obstructive Pulmonary Disease?

Background: Chronic obstructive pulmonary disease (COPD) has been frequently associated with frailty. The association between frailty and mortality in patients with COPD has not yet been fully elucidated and it remains controversial whether frailty or airflow limitation is more important in predicting mortality. Methods: A total of 141 subjects with stable COPD completed pulmonary function tests and the Kihon Checklist at baseline between 2015 and 2022 and were followed for a maximum of 95 months. Using the Kihon Checklist Total score, we classified patients' frailty status as robust (0-3), pre-frail (4-7), and frail (8-25). Results: At baseline, there were 67 (47.5%) in the robust group, 36 (25.5%) pre-frail, and 38 (27.0%) frail. Death was confirmed in 29 (20.5%). Univariate Cox proportional hazards analyses revealed that all predictive relationships of frailty and airflow limitation with mortality were statistically significant, and the C-index was similar, ranging from 0.63 to 0.66. According to the Log rank test and the Cox regression model, there was a significant difference between the frail and robust groups, (p=0.004 and p=0.005, respectively). No significant differences were found in either comparison with the pre-frail group. When stratification of frailty and FEV1 as well as sex were included as explanatory variables, multivariate Cox regression analysis showed a significant difference between the robust and frail groups with respect to mortality when the robust group was used as the reference (p=0.024). The robust and pre-frail groups did not differ significantly (p=0.163). Conclusion: The mortality of frail COPD patients is higher than those classed as robust. Despite frailty being a substantial mortality predictor, it is uncertain whether or not it is a better predictor than FEV1.

The Conceptual Independence of Health Status, Respiratory Symptoms and Dyspnea in Chronic Obstructive Pulmonary Disease in Real Clinical Practice.

The hypothesis that health status is the highest ranking concept, followed by respiratory symptoms and dyspnea as the lowest ranking concepts in subjects with chronic obstructive pulmonary disease (COPD) was tested in a real clinical setting with 157 subjects with stable COPD. Spearman's rank correlation coefficients for scores of health status using the COPD Assessment Test (CAT), respiratory symptoms using the COPD Evaluating Respiratory Symptoms (E-RS) and dyspnea using Dyspnea-12 (D-12) between any two were 0.6 to 0.7. Upon categorizing the patients as "abnormal" or "normal" according to the threshold, it was found that 30 patients (19.1%) had dyspnea, respiratory symptoms and impaired health status. Dyspnea was considered an important part of respiratory symptoms, though seven patients had dyspnea but no respiratory symptoms. There were 10 patients who had respiratory symptoms without dyspnea but without health status problems. Furthermore, there were six patients who had both dyspnea and respiratory symptoms but whose health status was classified as fine. Thus, the hypothesis was correct in approximately 85% of cases.

Comparison of Predictive Properties between Tools of Patient-Reported Outcomes: Risk Prediction for Three Future Events in Subjects with COPD.

BACKGROUND: Patient-reported outcome (PRO) measures must be evaluated for their discriminatory, evaluative, and predictive properties. However, the predictive capability remains unclear. We aimed to examine the predictive properties of several PRO measures of all-cause mortality, acute exacerbation of chronic obstructive pulmonary disease (COPD), and associated hospitalization. METHODS: A total of 122 outpatients with stable COPD were prospectively recruited and completed six self-administered paper questionnaires: the COPD Assessment Test (CAT), St. George's Respiratory Questionnaire (SGRQ), Baseline Dyspnea Index (BDI), Dyspnoea-12, Evaluating Respiratory Symptoms in COPD and Hyland Scale at baseline. Cox proportional hazards analyses were conducted to examine the relationships with future outcomes. RESULTS: A total of 66 patients experienced exacerbation, 41 were hospitalized, and 18 died. BDI, SGRQ Total and Activity, and CAT and Hyland Scale scores were significantly related to mortality (hazard ratio = 0.777, 1.027, 1.027, 1.077, and 0.951, respectively). The Hyland Scale score had the best predictive ability for PRO measures, but the C index did not reach the level of the most commonly used FEV1. Almost all clinical, physiological, and PRO measurements obtained at baseline were significant predictors of the first exacerbation and the first hospitalization due to it, with a few exceptions. CONCLUSIONS: Measurement of health status and the global scale of quality of life as well as some tools to assess breathlessness, were significant predictors of all-cause mortality, but their predictive capacity did not reach that of FEV1. In contrast, almost all baseline measurements were unexpectedly related to exacerbation and associated hospitalization.

Are Fatigue and Pain Overlooked in Subjects with Stable Chronic Obstructive Pulmonary Disease?

Although there have been many published reports on fatigue and pain in patients with chronic obstructive pulmonary disease (COPD), it is considered that these symptoms are seldom, if ever, asked about during consultations in Japanese clinical practice. To bridge this gap between the literature and daily clinical experience, the authors attempted to gain a better understanding of fatigue and pain in Japanese subjects with COPD. The Brief Fatigue Inventory (BFI) to analyse and quantify the degree of fatigue, the revised Short-Form McGill Pain Questionnaire 2 (SF-MPQ-2) for measuring pain and the Kihon Checklist to judge whether a participant is frail and elderly were administered to 89 subjects with stable COPD. The median BFI and SF-MPQ-2 Total scores were 1.00 [IQR: 0.11-2.78] and 0.00 [IQR: 0.00-0.27], respectively. They were all skewed toward the milder end of the respective scales. A floor effect was noted in around a quarter on the BFI and over half on the SF-MPQ-2. The BFI scores were significantly different between groups regarding frailty determined by the Kihon Checklist but not between groups classified by the severity of airflow limitation. Compared to the literature, neither fatigue nor pain are considered to be frequent, important problems in a real-world Japanese clinical setting, especially among subjects with mild to moderate COPD. In addition, our results might suggest that fatigue is more closely related to frailty than COPD.

Comparison between tools for measuring breathlessness: Cross-sectional validation of the Japanese version of the Dyspnoea-12.

INTRODUCTION: The Dyspnoea-12 (D-12) is a brief, easy to complete questionnaire for measuring breathlessness. OBJECTIVES: To facilitate further efforts to measure dyspnoea in real clinical settings, the authors aimed to develop and validate a Japanese version of the D-12 and also compare the D-12 with the Baseline Dyspnea Index (BDI) and the Activity component of the St. George's Respiratory Questionnaire (SGRQ). METHODS: The standardized procedure in accordance with international guidelines was used to create the translation. A validation study with a cross-sectional observational design was conducted on 122 subjects with stable chronic obstructive pulmonary disease (COPD). RESULTS: The internal consistency of the D-12 was high (Cronbach's coefficient α = 0.883) and similar to that of the BDI (α = 0.824) and SGRQ Activity (α = 0.872). The relationships between tools were statistically significant (|Rs | = 0.53 to 0.66). Although the scores obtained from all three tools were skewed toward the milder end of the respective scales, this deviation was most prominent in the D-12 with a floor effect of 48.4%. CONCLUSION: The Japanese version of the D-12 was successfully validated, but we should be careful of any floor effect and marked skew to the mild end of the scale, especially in subjects with mild COPD.

Is Blood Eosinophil Count a Biomarker for Chronic Obstructive Pulmonary Disease in a Real-World Clinical Setting? Predictive Property and Longitudinal Stability in Japanese Patients.

The authors examined predictive properties and the longitudinal stability of blood eosinophil count (BEC) or three strata (<100 cells/mm3, 100-299 cells/mm3 and ≥300 cells/mm3) in patients with chronic obstructive pulmonary disease (COPD) for up to six and a half years as part of a hospital-based cohort study. Of the 135 patients enrolled, 21 (15.6%) were confirmed to have died during the follow-up period. Episodes of acute exacerbation of COPD (AECOPD) were identified in 74 out of 130 available patients (56.9%), and admission due to AECOPD in 35 out of 132 (26.5%). Univariate Cox proportional hazards analyses revealed that almost all the age, forced expiratory volume in 1 s (FEV1) and health status measures using St. George's Respiratory Questionnaire (SGRQ) Total and COPD Assessment Test (CAT) Score were significantly related to these types of events, but the relationship between age and AECOPD did not reach statistical significance (p = 0.05). Neither BEC nor the three different groups stratified by BEC were significant predictors of any subsequent events. There were no significant differences in the BEC between Visits 1-3 (p = 0.127, Friedman test). The ICC value was 0.755 using log-transformed data, indicating excellent repeatability. In the case of assigning to strata, Fleiss' kappa was calculated to be 0.464, indicating moderate agreement. The predictive properties of BEC may be limited in a real-world Japanese clinical setting. Attention must be paid to the fact that the longitudinal stability of the three strata is regarded as moderate.

Are frailty and patient-reported outcomes independent in subjects with asthma? A cross-sectional observational study.

INTRODUCTION: This study examined the possible associations between frailty and patient-reported outcomes (PROs) in elderly patients with asthma. METHODS: Participants completed the Kihon Checklist for frailty screening as well as the following tools for measuring generic- and disease-specific health-related quality of life (HRQOL) and asthma control; the Medical Outcomes Study 36-item short form (SF-36), the Hyland Scale (global scale), the Asthma Quality of Life Questionnaire (AQLQ), the Asthma Control Test (ACT), and the Asthma Control Questionnaire (ACQ). RESULTS: Of 69 consecutive outpatients with asthma, 38 (55.1%), 21 (30.4%), and 10 (14.5%) were classified as robust, pre-frail, and frail, respectively. Eight out of 52 patients with asthma in the elderly (AIE) (>65 years old) (15.4%) were considered as being frail. The Kihon Checklist total score was significantly correlated with all the scores obtained from the SF-36, Hyland Scale, AQLQ, ACT, and ACQ. All these scores were significantly different between groups with and without frailty. From the viewpoint of correlation coefficient, SF-36 Physical Functioning correlated most strongly with a coefficient of -0.68 (P < .01), and the Hyland Scale score was second (RS = -0.46, P < .01). The correlations between the Kihon Checklist total score and lung function parameters were weak or negative (|RS | < 0.35). DISCUSSION: There were significant associations between frailty and PROs, particularly generic perception of HRQOL. Since the Kihon Checklist and PROs such as the HRQOL overlap somewhat in their evaluation of the patients' condition, there might be some similarities in the conceptual frameworks of frailty and quality of life.

The release of phosphorylated-HSP27 from activated platelets of obstructive sleep apnea syndrome (OSAS) patients.

BACKGROUND: Obstructive sleep apnea syndrome (OSAS) is a well known risk of arterial thrombosis that results in cardiovascular morbidity. It has been reported that platelet aggregability is enhanced in patients with OSAS. In the present study, we investigated whether phosphorylated-HSP27 is released from the activated platelets of OSAS patients. METHODS: Patients diagnosed with OSAS (n = 21) were recruited, and platelet-rich plasma (PRP) was stimulated by ADP, ristosetin, collagen, and thrombin receptor-activating peptide. Platelet aggregation was measured using an aggregometer with a laser-scattering system. The levels of protein phosphorylation and the released levels of phosphorylated-HSP27 were determined by Western blot analysis and an ELISA, respectively. RESULTS: The phosphorylation of HSP27 in the platelets was induced by the stimulators. The released levels of phosphorylated-HSP27 was correlated with the levels of phosphorylated-HSP27 stimulated by ADP or collagen. The levels of ADP-induced phosphorylated-HSP27 were correlated with those of both phosphorylated-protein kinase B (Akt) and phosphorylatd-p38 mitogen-activated protein kinase; however, the levels of phosphorylated-HSP27 stimulated by collagen were correlated with phosphorylated-Akt levels only. The ED50 value of ADP on the platelet aggregation in OSAS (1.067 ± 0.128 µM) was lower than that in healthy subjects (1.778 ± 0.122 µM) and was inversely correlated with both the value of minimum SpO2 and the released level of phosphorylated-HSP27 stimulated by ADP. CONCLUSION: The results strongly suggest that phosphorylated-HSP27 is released from the activated platelets of OSAS patients.

Comparison between electronic and paper versions of patient-reported outcome measures in subjects with chronic obstructive pulmonary disease: an observational study with a cross-over administration.

OBJECTIVES: A wide range of electronic devices can be used for data collection of patient-reported outcome (PRO) measures in subjects with chronic obstructive pulmonary disease (COPD). Although comparisons between electronic and paper-based PRO measures have been undertaken in asthmatics, it is currently uncertain whether electronic questionnaires work equally as well as paper versions in elderly subjects with COPD. The aim of this study was to compare the responses to paper and electronic versions of the Evaluating Respiratory Symptoms in COPD (E-RS) and the COPD Assessment Test (CAT). DESIGN: A randomised cross-over design was used to compare the responses to paper and electronic versions of the two tools. The interval between the two administrations was 1 week. SETTING: Electronic versions were self-administered under supervision using a tablet computer at our outpatient clinic (secondary care hospital in Japan) while paper questionnaires completed at home were requested to be returned by mail. It was intended that half of the patients completed the electronic versions of both questionnaires first, followed by the paper versions while the other half completed the paper versions first. PARTICIPANTS: Eighty-one subjects with stable COPD were included. RESULTS: The E-RS total scores (possible range 0-40) were 6.8±7.4 and 5.0±6.6 in the paper-based and electronic versions, respectively, and the CAT scores (possible range 0-40) were 10.0±7.4 and 8.6±7.8. In both questionnaires, higher scores indicate worse status. The relationship between electronic and paper versions showed significant reliability for both the E-RS total score and CAT score (intraclass correlation coefficient=0.82 and 0.89, respectively; both p<0.001). However, both the E-RS total and CAT scores were significantly higher in the paper versions (p<0.05). CONCLUSIONS: In both cases, the two versions of the same questionnaire cannot be used interchangeably even though they have both been validated.

Comparison of patient-reported outcomes during acute exacerbations of chronic obstructive pulmonary disease.

INTRODUCTION: The aim of this study was to investigate which patient-reported outcome measure was the best during the recovery phase from severe exacerbation of chronic obstructive pulmonary disease (COPD). METHODS: The Exacerbations of Chronic Pulmonary Disease Tool (EXACT), the COPD Assessment Test (CAT), the St George's Respiratory Questionnaire (SGRQ), the Dyspnoea-12 (D-12) and the Hyland Scale (global scale) were recorded every week for the first month and at 2 and 3 months in 33 hospitalised subjects with acute exacerbation of COPD (AECOPD). RESULTS: On the day of admission (day 1), the internal consistency of the EXACT total score was high (Cronbach's alpha coefficient=0.89). The EXACT total, CAT, SGRQ total and Hyland Scale scores obtained on day 1 appeared to be normally distributed. Neither floor nor ceiling effects were observed for the EXACT total and SGRQ total scores. The EXACT total score improved from 50.5±12.4 to 32.5±14.3, and the CAT score also improved from 24.4±8.5 to 13.5±8.4 during the first 2 weeks, and the effect sizes (ES) of the EXACT total and CAT score were -1.40 and -1.36, respectively. The SGRQ, Hyland Scale and D-12 were less responsive, with ES of -0.59, 0.96 and -0.90, respectively. DISCUSSION: The EXACT total and CAT scores are shown to be more responsive measures during the recovery phase from severe exacerbation. Considering the conceptual framework, it is recommended that the EXACT total score may be the best measure during the recovery phase from AECOPD. The reasons for the outstanding responsiveness of the CAT are still unknown.

Late-onset chest wall abscess due to a biodegradable rib pin infection after lung transplantation.

A 55-year-old man with end-stage emphysema underwent a right single-lung transplantation through a posterolateral thoracotomy. The fifth rib was divided and fused back using a biodegradable pin made of polylactide acid and hydroxyapatite. Two weeks postoperatively, he suffered from central vein catheter-related sepsis due to methicillin-sensitive Staphylococcus aureus. After being successfully treated for sepsis, he was discharged. However, 3 months later, computed tomography revealed multiple loculated abscesses in the chest wall and the right pleural space. Reoperative thoracotomy revealed abscesses mainly located around the fifth rib, where the pin was inserted. Both cultures of the abscess and the fifth rib were positive for methicillin-sensitive S. aureus, which suggested that the rib pin was the cause of the secondary infection. This case suggests the rib pins, even if they are biodegradable, could have a risk of infections side effect especially for the immunosuppressed patients.

Frailty and patient-reported outcomes in subjects with chronic obstructive pulmonary disease: are they independent entities?

INTRODUCTION: There is a hypothesis that chronic obstructive pulmonary disease (COPD) is an accelerated ageing disease. Frailty is a geriatric syndrome characterised by physical, psychological and social vulnerability, thought to be a feature of ageing. The authors aimed to explore the relationship between frailty and physiological and patient-reported outcomes (PROs) in subjects with stable COPD. METHODS: We administered the Kihon Checklist that has been validated for frailty screening. We also assessed patient-reported measurements of health status and dyspnoea using the COPD Assessment Test (CAT), the St. George's Respiratory Questionnaire (SGRQ), the Hyland Scale, the Medical Outcomes Study 36-item short-form (SF-36), the Baseline Dyspnea Index (BDI) and the Dyspnea-12 (D-12). Pulmonary function was also measured. RESULTS: Of 79 consecutive COPD outpatients, 38 (48.1%), 24 (30.4%) and 17 (21.5%) patients were classified as robust, prefrail and frail, respectively. The total Kihon Checklist score was significantly weakly to moderately correlated with the CAT score (Spearman's rank correlation coefficient (Rs)=0.38, p<0.01), the SGRQ total score (Rs=0.65, p<0.01), the Hyland Scale score (Rs=-0.54, p<0.01), all subscale scores of the SF-36 (Rs=-0.64 to -0.31, p<0.01), the BDI score (Rs=-0.46, p<0.01) and the D-12 score (Rs=0.41, p<0.01). We found no or only weak correlations between the total Kihon Checklist score and lung function measurements. We found statistically significant between-group (robust, prefrail and frail) differences in most PRO scores. Using stepwise multiple regression analyses to identify the variables that predicted the total Kihon Checklist score, the SGRQ total score alone significantly explained 49.1% of the variance (p<0.01). DISCUSSION: Frailty was significantly correlated with PROs, especially health status, unlike lung function. Frailty should be assessed in addition to PROs separately from lung function as part of multidimensional analyses of COPD.

A case of pulmonary actinomycosis diagnosed by transbronchial lung biopsy.

A previously healthy 73-year-old man was hospitalized with left complicated effusion and a consolidation in the left upper lung. He underwent a chest tube insertion and was treated with clindamycin but the consolidation remained after the treatment. We subsequently performed flexible bronchoscopy but it was impossible to make a diagnosis. Three months later, the consolidation had worsened so we performed another bronchoscopy. Finally, we were able to diagnose the consolidation as pulmonary actinomycosis, and to treat the condition appropriately. Pulmonary actinomycosis is a rare and difficult condition to diagnose. There are many conditions with similar clinical features, such as tuberculosis, fungal infections, lung abscesses, and lung malignancy. Respiratory physicians should consider the possibility of pulmonary actinomycosis when investigating patients with persistent pulmonary infiltrations. Early diagnosis and correct treatment may lead to a good prognosis and prevent unnecessary surgery.

Exogenous induction of unphosphorylated PTEN reduces TGFß-induced extracellular matrix expressions in lung fibroblasts.

Transforming growth factor ß (TGFß) plays an important role in regulating aberrant extracellular matrix (ECM) production from alveolar/epithelial cells (AECs) and fibroblasts in pulmonary fibrosis. Although the tumor suppressor gene phosphatase and tensin homologue deleted from chromosome 10 (PTEN) can negatively control many TGFß-activated signaling pathways via the phosphatase activity, hyperactivation of the TGFß-related signaling pathways is often observed in fibrosis. Loss of PTEN expression might cause TGFß-induced ECM production. In addition, TGFß was recently shown to induce loss of PTEN enzymatic activity by phosphorylating the PTEN C-terminus. Therefore, we hypothesized that exogenous transfer of unphosphorylated PTEN (PTEN4A) might lead to reduce TGFß-induced ECM expression in not only epithelial cells but also fibroblasts. Adenovirus-based exogenous PTEN4A induction successfully reduced TGFß-induced fibronectin expression and retained ß-catenin at the cell membrane in human epithelial cells. Exogenous unphosphorylated PTEN also attenuated TGFß-induced ECM production and inhibited TGFß-induced ß-catenin translocation in a human fibroblast cell line and in mouse primary isolated lung fibroblasts. Conversely, TGFß-induced α-smooth muscle actin expression did not seem to be inhibited in these fibroblasts. Our data suggest that exogenous administration of unphosphorylated PTEN might be a promising strategy to restore TGFß-induced loss of PTEN activity and reduce aberrant TGFß-induced ECM production from epithelial cells and fibroblasts in lung fibrosis as compared with wild-type PTEN induction.

Hypoxia-induced modulation of PTEN activity and EMT phenotypes in lung cancers.

BACKGROUND: Persistent hypoxia stimulation, one of the most critical microenvironmental factors, accelerates the acquisition of epithelial-mesenchymal transition (EMT) phenotypes in lung cancer cells. Loss of phosphatase and tensin homologue deleted from chromosome 10 (PTEN) expression might accelerate the development of lung cancer in vivo. Recent studies suggest that tumor microenvironmental factors might modulate the PTEN activity though a decrease in total PTEN expression and an increase in phosphorylation of the PTEN C-terminus (p-PTEN), resulting in the acquisition of the EMT phenotypes. Nevertheless, it is not known whether persistent hypoxia can modulate PTEN phosphatase activity or whether hypoxia-induced EMT phenotypes are negatively regulated by the PTEN phosphatase activity. We aimed to investigate hypoxia-induced modulation of PTEN activity and EMT phenotypes in lung cancers. METHODS: Western blotting was performed in five lung cancer cell lines to evaluate total PTEN expression levels and the PTEN activation. In a xenograft model of lung cancer cells with endogenous PTEN expression, the PTEN expression was evaluated by immunohistochemistry. To examine the effect of hypoxia on phenotypic alterations in lung cancer cells in vitro, the cells were cultured under hypoxia. The effect of unphosphorylated PTEN (PTEN4A) induction on hypoxia-induced EMT phenotypes was evaluated, by using a Dox-dependent gene expression system. RESULTS: Lung cancer cells involving the EMT phenotypes showed a decrease in total PTEN expression and an increase in p-PTEN. In a xenograft model, loss of PTEN expression was observed in the tumor lesions showing tissue hypoxia. Persistent hypoxia yielded an approximately eight-fold increase in the p-PTEN/PTEN ratio in vitro. PTEN4A did not affect stabilization of hypoxia-inducible factor 1α. PTEN4A blunted hypoxia-induced EMT via inhibition of ß-catenin translocation into the cytoplasm and nucleus. CONCLUSION: Our study strengthens the therapeutic possibility that compensatory induction of unphosphorylated PTEN may inhibit the acquisition of EMT phenotypes in lung cancer cells under persistent hypoxia.

Direct regulation of transforming growth factor ß-induced epithelial-mesenchymal transition by the protein phosphatase activity of unphosphorylated PTEN in lung cancer cells.

Transforming growth factor ß (TGFß) causes the acquisition of epithelial-mesenchymal transition (EMT). Although the tumor suppressor gene PTEN (phosphatase and tensin homologue deleted from chromosome 10) can negatively regulate many signaling pathways activated by TGFß, hyperactivation of these signaling pathways is observed in lung cancer cells. We recently showed that PTEN might be subject to TGFß-induced phosphorylation of its C-terminus, resulting in a loss of its enzyme activities; PTEN with an unphosphorylated C-terminus (PTEN4A), but not PTEN wild, inhibits TGFß-induced EMT. Nevertheless, whether or not the blockade of TGFß-induced EMT by the PTEN phosphatase activity might be attributed to the unphosphorylated PTEN C-terminus itself has not been fully determined. Furthermore, the lipid phosphatase activity of PTEN is well characterized, whereas the protein phosphatase activity has not been determined. By using lung cancer cells carrying PTEN domain deletions or point mutants, we investigated the role of PTEN protein phosphatase activities on TGFß-induced EMT in lung cancer cells. The unphosphorylated PTEN C-terminus might not directly retain the phosphatase activities and repress TGFß-induced EMT; the modification that keeps the PTEN C-terminus not phosphorylated might enable PTEN to retain the phosphatase activity. PTEN4A with G129E mutation, which lacks lipid phosphatase activity but retains protein phosphatase activity, repressed TGFß-induced EMT. Furthermore, the protein phosphatase activity of PTEN4A depended on an essential association between the C2 and phosphatase domains. These data suggest that the protein phosphatase activity of PTEN with an unphosphorylated C-terminus might be a therapeutic target to negatively regulate TGFß-induced EMT in lung cancer cells.

Involvement of TGFß-induced phosphorylation of the PTEN C-terminus on TGFß-induced acquisition of malignant phenotypes in lung cancer cells.

Transforming growth factor ß (TGFß) derived from the tumor microenvironment induces malignant phenotypes such as epithelial-mesenchymal transition (EMT) and aberrant cell motility in lung cancers. TGFß-induced translocation of ß-catenin from E-cadherin complexes into the cytoplasm is involved in the transcription of EMT target genes. PTEN (phosphatase and tensin homologue deleted from chromosome 10) is known to exert phosphatase activity by binding to E-cadherin complexes via ß-catenin, and recent studies suggest that phosphorylation of the PTEN C-terminus tail might cause loss of this PTEN phosphatase activity. However, whether TGFß can modulate both ß-catenin translocation and PTEN phosphatase activity via phosphorylation of the PTEN C-terminus remains elusive. Furthermore, the role of phosphorylation of the PTEN C-terminus in TGFß-induced malignant phenotypes has not been evaluated. To investigate whether modulation of phosphorylation of the PTEN C-terminus can regulate malignant phenotypes, here we established lung cancer cells expressing PTEN protein with mutation of phosphorylation sites in the PTEN C-terminus (PTEN4A). We found that TGFß stimulation yielded a two-fold increase in the phosphorylated -PTEN/PTEN ratio. Expression of PTEN4A repressed TGFß-induced EMT and cell motility even after snail expression. Our data showed that PTEN4A might repress EMT through complete blockade of ß-catenin translocation into the cytoplasm, besides the inhibitory effect of PTEN4A on TGFß-induced activation of smad-independent signaling pathways. In a xenograft model, the tumor growth ratio was repressed in cells expressing PTEN4A. Taken together, these data suggest that phosphorylation sites in the PTEN C-terminus might be a therapeutic target for TGFß-induced malignant phenotypes in lung cancer cells.

Differential modulation of surfactant protein D under acute and persistent hypoxia in acute lung injury.

Hypoxia contributes to the development of fibrosis with epithelial-mesenchymal transition (EMT) via stimulation of hypoxia-inducible factor 1α (HIF-1α) and de novo twist expression. Although hypoxemia is associated with increasing levels of surfactant protein D (SP-D) in acute lung injury (ALI), the longitudinal effects of hypoxia on SP-D expression in lung tissue injury/fibrosis have not been fully evaluated. Here, the involvement of hypoxia and SP-D modulation was evaluated in a model of bleomycin-induced lung injury. We also investigated the molecular mechanisms by which hypoxia might modulate SP-D expression in alveolar cells, by using a doxycycline (Dox)-dependent HIF-1α expression system. Tissue hypoxia and altered SP-D levels were present in bleomycin-induced fibrotic lesions. Acute hypoxia induced SP-D expression, supported by the finding that Dox-induced expression of HIF-1α increased SP-D expression. In contrast, persistent hypoxia repressed SP-D expression coupled with an EMT phenotype and twist expression. Long-term expression of HIF-1α caused SP-D repression with twist expression. Ectopic twist expression repressed SP-D expression. The longitudinal observation of hypoxia and SP-D levels in ALI in vivo was supported by the finding that HIF-1α expression stabilized by acute hypoxia induced increasing SP-D expression in alveolar cells, whereas persistent hypoxia induced de novo twist expression in these cells, causing repression of SP-D and acquisition of an EMT phenotype. Thus this is the first study to demonstrate the molecular mechanisms, in which SP-D expression under acute and persistent hypoxia in acute lung injury might be differentially modulated by stabilized HIF-1α expression and de novo twist expression.

Involvement of the transcription factor twist in phenotype alteration through epithelial-mesenchymal transition in lung cancer cells.

Epithelial-mesenchymal transition (EMT), which involves the persistent loss of epithelial markers and expression of mesenchymal markers, is assumed to have a critical role in not only tissue development during embryogenesis but also central mechanisms that enhance the invasive and metastatic ability of cancer cells. Twist has been identified to play an essential role in EMT-mediated tumor invasion and metastasis. Although recent studies suggest that twist expression levels in tissue specimens of lung cancer might be associated with prognosis, the expression of twist in lung cancer cells itself and its effect have not been fully evaluated. Here, we evaluated twist expression and its effect on phenotype alteration in lung cancer cell lines. Twist expression varied among human lung cancer cell lines. The lung cancer cell lines with high twist expression also tended to show a high vimentin/E-cadherin ratio, which was supported by a migration assay, in which high twist expression gave rise to high cell motility. Furthermore, in comparison to control cells, the lung cancer cells with ectopic expression of twist showed a significant phenotype alteration through EMT and an increasing ability to migrate in vitro, in part, due to a tenfold increase in matrix metalloproteinases activity and almost a 60% increase in modulation of focal adhesion kinase activity, although a contribution of microRNA appeared unlikely in our study. Our present analysis of twist expression in lung cancer provide clues to comprehensive understanding of the mechanisms, by which metastasis often develops in lung cancer.