Cell-line dependency in cerebral organoid induction: cautionary observations in Alzheimer's disease patient-derived induced pluripotent stem cells.

The cerebral organoid (CO) model has been used in the study of various neurodegenerative diseases owing to its physiological implications. However, the CO model may only be representative of certain clinical findings in affected patients, while some features are not recapitulated. In this study, we found that neurons in the CO model from patients with Alzheimer's disease were less responsive to depolarization, in contrast to previous reports. This difference may be partly attributed to the variations in brain spatial identity depending on the genetic background of the induced pluripotent stem cells. Our current observation raises concerns that the phenotypes observed in the CO model need to be carefully evaluated for their clinical implications.

Respiratory sound classification for crackles, wheezes, and rhonchi in the clinical field using deep learning.

Auscultation has been essential part of the physical examination; this is non-invasive, real-time, and very informative. Detection of abnormal respiratory sounds with a stethoscope is important in diagnosing respiratory diseases and providing first aid. However, accurate interpretation of respiratory sounds requires clinician's considerable expertise, so trainees such as interns and residents sometimes misidentify respiratory sounds. To overcome such limitations, we tried to develop an automated classification of breath sounds. We utilized deep learning convolutional neural network (CNN) to categorize 1918 respiratory sounds (normal, crackles, wheezes, rhonchi) recorded in the clinical setting. We developed the predictive model for respiratory sound classification combining pretrained image feature extractor of series, respiratory sound, and CNN classifier. It detected abnormal sounds with an accuracy of 86.5% and the area under the ROC curve (AUC) of 0.93. It further classified abnormal lung sounds into crackles, wheezes, or rhonchi with an overall accuracy of 85.7% and a mean AUC of 0.92. On the other hand, as a result of respiratory sound classification by different groups showed varying degree in terms of accuracy; the overall accuracies were 60.3% for medical students, 53.4% for interns, 68.8% for residents, and 80.1% for fellows. Our deep learning-based classification would be able to complement the inaccuracies of clinicians' auscultation, and it may aid in the rapid diagnosis and appropriate treatment of respiratory diseases.

New Insights into the Clinical Implications of Yes-Associated Protein in Lung Cancer: Roles in Drug Resistance, Tumor Immunity, Autophagy, and Organoid Development.

Despite significant innovations in lung cancer treatment, such as targeted therapy and immunotherapy, lung cancer is still the principal cause of cancer-associated death. Novel strategies to overcome drug resistance and inhibit metastasis in cancer are urgently needed. The Hippo pathway and its effector, Yes-associated protein (YAP), play crucial roles in lung development and alveolar differentiation. YAP is known to mediate mechanotransduction, an important process in lung homeostasis and fibrosis. In lung cancer, YAP promotes metastasis and confers resistance against chemotherapeutic drugs and targeted agents. Recent studies revealed that YAP directly controls the expression of programmed death-ligand 1 (PD-L1) and modulates the tumor microenvironment (TME). YAP not only has a profound relationship with autophagy in lung cancer but also controls alveolar differentiation, and is responsible for tubular structure formation in lung organoids. In this review, we discuss the various roles and clinical implications of YAP in lung cancer and propose that targeting YAP can be a promising strategy for treating lung cancer.

Spontaneous regression of incidentally diagnosed bronchial squamous cell lung carcinoma after severe bronchitis: A case report.

Spontaneous regression of lung cancer is exceptionally rare. But there have been several intriguing cases reported in early and even advanced stages of lung cancer. Although the exact mechanism remains to be elucidated, the inflammation and immunologic response have been suggested as one of the means of spontaneous regression. Chronic inflammation is generally known to induce and aggravate tumorigenesis, but the relationship between cancer and inflammation highly depends on the contexts. Here, we present a case of a 60-year-old male ex-smoker who complained of recurrent hemoptysis, cough, and purulent sputum. The initial chest CT scan revealed diffuse bronchial thickening and an endobronchial mass-like lesion in the left lingular segment. The bronchoscopic and pathological findings also suggested a diagnosis of squamous cell carcinoma with severe mucosal inflammation. He was treated with antibiotics for the bronchitis during the first 1 week and his symptoms markedly improved. After 3 weeks, he underwent a follow-up examination. Chest computed tomography and bronchoscopy revealed the significant improvement of the bronchial narrowing and mucosal edema. Biopsy was performed several times around the lesion where the tissue was initially taken. However, the pathological results showed only chronic inflammation of bronchi, not cancer cells. Fortunately, there was no recurrence of lung cancer in follow-up chest computed tomography or bronchoscopy for almost 5 years. In this case, the incidentally diagnosed bronchial squamous cell carcinoma disappeared after severe inflammatory reaction of the bronchial wall. The clinician should remind the risk of early lung cancer accompanied with bronchitis in high-risk patients of lung cancer and also be aware that although it is very rare, the lesions could spontaneously regress.

Targeting YAP-p62 signaling axis suppresses the EGFR-TKI-resistant lung adenocarcinoma.

BACKGROUND: Despite the progress of advanced target therapeutic agents and immune checkpoint inhibitors, EGFR-TKI resistance is still one of the biggest obstacles in treating lung cancer. Clinical studies with autophagy inhibitors are actively underway to overcome drug resistance. METHODS: We used PC9, PC9/GR, and HCC827/GR cell lines to evaluate the activation of autophagy and EGFR-TKI resistance. Chloroquine was applied as an autophagic blocker and verteporfin was utilized as a YAP inhibitor. RESULTS: In this study, we tried to reveal the effect of autophagy adaptor p62 which is accumulated by autophagy inhibitor in EGFR-TKI-resistant lung adenocarcinoma. We identified that p62 has oncogenic functions that induce cell proliferation and invasion of EGFR-TKI-resistant lung adenocarcinoma. Interestingly, we found for the first time that YAP regulates p62 transcription through ERK, and YAP inhibition can suppress the expression of oncogenic p62. We also confirmed that the expressions of p62 and YAP have a positive correlation in EGFR-mutant lung adenocarcinoma patients. To block cell survival via perturbing YAP-p62 axis, we treated EGFR-TKI-resistant lung cancer cells with YAP inhibitor verteporfin. Remarkably, verteporfin effectively caused the death of EGFR-TKI-resistant lung cancer cells by decreasing the expressions of p62 with oncogenic function, YAP, and its target PD-L1. So, the cumulative effect of oncogenic p62 should be considered when using autophagy inhibitors, especially drugs that act at the last stage of autophagy such as chloroquine and bafilomycin A1. CONCLUSION: Finally, we suggest that targeting YAP-p62 signaling axis can be useful to suppress the EGFR-TKI-resistant lung cancer. Therefore, drug repurposing of verteporfin for lung cancer treatment may be valuable to consider because it can inhibit critical targets: p62, YAP, and PD-L1 at the same time.

Suppression of Mig-6 overcomes the acquired EGFR-TKI resistance of lung adenocarcinoma.

BACKGROUND: The resistance of lung cancer to epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) is one of the unconquered frontiers in chemotherapy. Mitogen-inducible gene 6 (Mig-6) is known to inhibit the kinase activity of epidermal growth factor receptor (EGFR). Similarly, numerous studies of mouse models suggested tumor suppressive function of Mig-6 in lung cancer. On the contrary, the results of clinical investigations revealed that lung cancer patients with elevated expression of Mig-6 are associated with a poor prognosis. More recent work showed that unlike wild type (WT) EGFR, mutant EGFR phosphorylates Mig-6 and phosphorylated Mig-6 negatively regulates the degradation of EGFR mutants in lung adenocarcinoma. Here, we tried to untangle the controversies surrounding Mig-6 function as a protagonist or an antagonist of EGFR-TKI resistant lung cancer. METHODS: We compared the expression and phosphorylation status of Mig-6 in the EGFR-TKI resistant lung adenocarcinoma (PC9/GR cells) to EGFR-TKI sensitive lung adenocarcinoma (PC9 cells). We investigated the function of Mig-6 by either depletion or overexpression of Mig-6 in those cells and evaluated the efficacy of combining of Mig-6 knock-down and EGFR-TKI treatment in PC9/GR. The correlation between Mig-6 expressions and the prognoses of lung adenocarcinoma was examined by The Cancer Genome Atlas (TCGA) data and clinical samples. RESULTS: Our results indicated that the expression of Mig-6 was significantly increased in PC9/GR cells compared to that of PC9 cells. The significant portion of Mig-6 existed as a phosphorylated form in PC9 and PC9/GR cells. Moreover, overexpression of Mig-6 significantly increased the cell proliferation, invasion and epithelial mesenchymal transition (EMT) in PC9 cells. Combination of Mig-6 knock-down and EGFR-TKI treatment significantly overcame the EGFR-TKI resistance of PC9/GR cells. In addition, our analyses of clinical samples confirmed that high Mig-6 expressions positively correlate with a poor prognosis and EGFR-TKI resistance in lung adenocarcinoma. CONCLUSION: Our findings reinforce scientific notion of Mig-6 as an oncoprotein in the context of EGFR-TKI resistant lung adenocarcinoma. We propose that targeting Mig-6 may be a promising strategy to overcome the EGFR-TKI resistance in lung cancer.

Mass spectrometric investigation of concentration-dependent effect of curcumin and oxidative stress on intracellular glutathione levels.

Herein, we investigated the correlation between curcumin and glutathione (GSH) levels in mammalian cells using gold nanoparticles (AuNPs) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). GSH exists in high concentration in the cytosol and acts as a major antioxidant and reducing agent in organisms. Previous studies showed that curcumin, a well-known antioxidant with anti-inflammatory, anti-proliferative, and anti-carcinogenic activities, affects GSH levels in mammalian cells. However, the correlation between their levels remains controversial and has not yet been completely elucidated. This study used our recent strategy of GSH quantification, where GSH in cell lysate is captured on maleimide groups of AuNPs and analyzed using MALDI-TOF MS with isotopomer GSH (GSH*)-conjugated AuNPs as an internal standard. The comparison between GSH and GSH* relative intensities allows the quantitation of GSH in cells. In this way, GSH levels in mammalian cells were investigated after incubation with curcumin at various concentrations with or without oxidative stress. We observed that intracellular GSH levels were affected by curcumin in a concentration-dependent manner with oxidative stress; GSH levels decrease at a lower curcumin concentration, which can be recovered at increased curcumin concentrations. We also found that the GSH level increased at all curcumin concentrations after a certain incubation time. We believe our strategy can be commonly used to determine GSH levels in cells that are treated differently with various exogenous stimulants like reactive oxygen species, biofunctional natural products, and drug candidates. Graphical abstract.

Hippo effector YAP directly regulates the expression of PD-L1 transcripts in EGFR-TKI-resistant lung adenocarcinoma.

Developments of EGFR-TKI and immunotherapy targeting the PD1/PD-L1 pathway are considered most important medical breakthroughs in lung cancer treatment. Nowadays, 3rd generation EGFR TKI is widely used for T790M positive 1st and 2nd EGFR-TKI resistant lung cancer patients. Immunotherapy is powerful option for lung cancer patients without drug targets and chemotherapy resistant patients. It also has changed the concept of conventional anti-cancer therapy in the point of regulating tumor microenvironment. There are many studies linking these two important pathways. Recent studies demonstrated that PD-L1 expression is significantly correlated to the mutation status of EGFR, and activation of EGFR signaling can also induce the expression of PD-L1. However, the real linker between PD-L1 and EGFR signaling remains to be revealed. Our previous study revealed that the Hippo pathway effector YAP confers EGFR-TKI resistance in lung adenocarcinoma, and inhibition of YAP restores sensitivity to EGFR-TKIs. Thus, we examined whether PD-L1 is relevant, in terms of conferring EGFR-TKI resistance and whether YAP directly regulates the expression of PD-L1 in this context. First, we compared the expression levels of PD-L1 and YAP between EGFR-TKI-resistant PC9 cells and the parental PC9 adenocarcinoma cells. The expression levels of both YAP and PD-L1 were markedly higher in the EGFR-TKI-resistant cells compared to the parental cells, suggesting differential expression pattern between two cell types. YAP knockdown significantly decreased the expression of PD-L1 in the EGFR-TKI-resistant cells, while YAP overexpression increased the expression of PD-L1 in the parental PC9 cells. Then, our results revealed that YAP regulates the transcription of PD-L1, and the YAP/TEAD complex binds to the PD-L1 promoter. Surprisingly, knockdown of PD-L1 was sufficient to decrease cell proliferation and wound healing in the EGFR-TKI-resistant PC9 cells. These data suggest a PD1-independent oncogenic function of PD-L1. The Hippo effector YAP plays a crucial role in linking the PD-L1 and EGFR-TKI resistance by directly regulating the expression of PD-L1 in lung cancer. Targeting PD-L1 directly or via YAP could provide an effective therapeutic strategy for EGFR-TKI-resistant lung adenocarcinoma.

The novel YAP target gene, SGK1, upregulates TAZ activity by blocking GSK3ß-mediated TAZ destabilization.

YAP (Yes-associated protein) and TAZ (transcription activator with PDZ binding motif) are important in tissue regeneration and cancer development, highlighting the importance of discovering partners that regulate their oncogenicity. SGK1 (serum/glucocorticoid regulated kinase 1), initially identified as a homolog of Akt in phosphoinositide 3-kinase signaling, acts as a serine/threonine protein kinase in multiple oncogenic pathways. However, possible links between SGK1 and Hippo-YAP/TAZ signaling remain unexplored. Here, we reveal that SGK1 is a potential positive feedback regulator of YAP and TAZ, showing that the TEAD-YAP/TAZ complex directly activates SGK1 transcription by binding to the distal enhancer of SGK1, and SGK1, in turn, stabilizes YAP/TAZ. Moreover, we demonstrate that expression of YAP/TAZ target genes is positively regulated by SGK1. Mechanistically, SGK1 inhibits ubiquitin-mediated degradation of TAZ by inhibiting GSK3ß activity. These findings expand our understanding of YAP/TAZ regulation to include the novel downstream target of YAP, SGK1.

The E3 ubiquitin ligase CHIP selectively regulates mutant epidermal growth factor receptor by ubiquitination and degradation.

Somatic mutation in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) is a decisive factor for the therapeutic response to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in lung adenocarcinoma. The stability of mutant EGFR is maintained by various regulators, including heat shock protein 90 (Hsp90). The C terminus of Hsc70-interacting protein (CHIP) is a Hsp70/Hsp90 co-chaperone and exhibits E3 ubiquitin ligase activity. The high-affinity Hsp90-CHIP complex recognizes and selectively regulates their client proteins. CHIP also works with its own E3 ligase activity independently of Hsp70/Hsp90. Here, we investigated the role of CHIP in regulating EGFR in lung adenocarcinoma and also evaluated the specificity of CHIP's effects on mutant EGFR. In HEK 293T cells transfected with either WT EGFR or EGFR mutants, the overexpression of CHIP selectively decreased the expression of certain EGFR mutants (G719S, L747_E749del A750P and L858R) but not WT EGFR. In a pull-down assay, CHIP selectively interacted with EGFR mutants and simultaneously induced their ubiquitination and proteasomal degradation. The expressions of mutant EGFR in PC9 and H1975 were diminished by CHIP, while the expression of WT EGFR in A549 was nearly not affected. In addition, CHIP overexpression inhibited cell proliferation and xenograft's tumor growth of EGFR mutant cell lines, but not WT EGFR cell lines. EGFR mutant specific ubiquitination by CHIP may provide a crucial regulating mechanism for EGFR in lung adenocarcinoma. Our results suggest that CHIP can be novel therapeutic target for overcoming the EGFR TKI resistance.

Hippo pathway effector YAP inhibition restores the sensitivity of EGFR-TKI in lung adenocarcinoma having primary or acquired EGFR-TKI resistance.

The efficacy of EGFR-tyrosine kinase inhibitors (TKIs) is significantly limited by various resistance mechanisms to those drugs. The resistance to EGFR-TKI is largely divided by two classes; acquired resistance after EGFR-TKI treatment, and primary resistance marked by cancer cell's dependence on other oncogene, such as KRAS. YAP has emerged as critical oncogene in conferring drug resistance against targeted therapy. In this study, we evaluated the role of YAP in primary and acquired EGFR-TKI resistance using gefitinib-resistant A549 and PC9 cells and their parental cell lines. Our study revealed that EGFR-TKI resistance is associated with enhanced YAP activity. Notably, YAP activation was independent of the Hippo pathway. We confirmed that AXL is a downstream target of YAP that confers EGFR-TKI resistance. And our results showed that YAP can induce ERK activation in lung adenocarcinoma. The combination of YAP inhibition with EGFR-TKI overcomes primary and acquired EGFR-TKI resistance. We also found increased YAP expression in human lung cancer after acquiring EGFR-TKI resistance. Collectively, we suggest a novel EGFR-TKI resistance mechanism involving YAP activation and suggest targeting YAP and EGFR simultaneously may be a breakthrough treatment of primary and acquired EGFR-TKI resistant lung cancer.

Hippo-Foxa2 signaling pathway plays a role in peripheral lung maturation and surfactant homeostasis.

Respiratory distress syndrome (RDS), which is induced by insufficient production of surfactant, is the leading cause of mortality in preterm babies. Although several transcription factors are known to be involved in surfactant protein expression, the molecular mechanisms and signaling pathways upstream of these transcription factors have remained elusive. Here, using mammalian Hippo kinases (Mst1/2, mammalian sterile 20-like kinase 1/2) conditional knockout mice, we demonstrate that Mst1/2 kinases are critical for orchestration of transcription factors involved in surfactant protein homeostasis and prevention of RDS. Mice lacking Mst1/2 in the respiratory epithelium exhibited perinatal mortality with respiratory failure and their lungs contained fewer type I pneumocytes and more immature type II pneumocytes lacking microvilli, lamellar bodies, and surfactant protein expression, pointing to peripheral lung immaturity and RDS. In contrast to previous findings of YAP (Yes-associated protein)-mediated canonical Hippo signaling in the liver and intestine, loss of Mst1/2 kinases induced the defects in pneumocyte differentiation independently of YAP hyperactivity. We instead found that Mst1/2 kinases stabilized and phosphorylated the transcription factor Foxa2 (forkhead box A2), which regulates pneumocyte maturation and surfactant protein expression. Taken together, our results suggest that the mammalian Hippo kinases play crucial roles in surfactant homeostasis and coordination of peripheral lung differentiation through regulation of Foxa2 rather than of YAP.

A novel mutation W252X in the WAS gene in a Korean patient with Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency disorder characterized by recurrent infection, eczema, and microthrombocytopenia. WAS is inherited in an X-linked recessive pattern, and various mutations in the WAS gene on the X chromosome are the genetic basis of WAS. A 7-month-old Korean boy presented with recurrent bloody diarrhea, eczema, and persistent thrombocytopenia with small platelets. Direct sequence analysis of the entire coding region of the WAS gene showed a novel nonsense mutation with a G-to-A substitution at the nucleotide position 756 on exon 8, leading to a premature termination at codon 252 (c.756G>A; p.W252X). Family study revealed that neither of the parents had the mutation, indicating the de novo occurrence of the mutation.