BRAFV600E promotes anchorage-independent growth but inhibits anchorage-dependent growth in hTERT/Cdk4-Immortalized normal human bronchial epithelial cells.

Certain oncogenes, including mutant RAS and BRAF, induce a type of senescence known as oncogene-induced senescence (OIS) in normal cells in a cell-type-specific manner. OIS serves as a barrier to transformation by activated oncogenes. Our previous studies showed that mutant KRASV12 did not efficiently induce OIS in an hTERT/Cdk4-immortalized normal human bronchial epithelial cell line (HBEC3), but it did enhance both anchorage-dependent and anchorage-independent growth. In this study, we investigated whether mutant BRAF, a well-known inducer of OIS, could trigger OIS in HBEC3 cells. We also assessed the impact of mutant BRAF on the growth of HBEC3 cells, as no previous studies have examined this using a normal bronchial epithelial cell line model. We established an HBEC3 cell line, designated as HBEC3-BIN, that expresses mutant BRAFV600E in a doxycycline-regulated manner. Unlike our previous finding that KRASV12 upregulated both pERK and pAKT, mutant BRAFV600E upregulated pERK but not pAKT in HBEC3-BIN cells. Similar to KRASV12, BRAFV600E did not efficiently induce OIS. Interestingly, while BRAFV600E inhibited colony formation in anchorage-dependent conditions, it dramatically enhanced colony formation in anchorage-independent conditions in HBEC3-BIN. In HBEC3 cells without BRAFV600E or KRASV12 expression, p21 was only detected in the cytoplasm, and its localization was not altered by the expression of BRAFV600E or KRASV12. Next-generation sequencing analysis revealed an enrichment of gene sets known to be involved in carcinogenesis, including IL3/JAK/STAT3, IL2, STAT5, and the EMT pathway. Our results indicate that, unlike KRASV12, which promoted both, BRAFV600E enhances anchorage-independent growth but inhibits anchorage-dependent growth of HBEC3. This contrast may result from differences in activation signaling in the downstream pathways. Furthermore, HBEC3 cells appear to be inherently resistant to OIS, which may be partly due to the fact that p21 remains localized in the cytoplasm upon expression of BRAFV600E or KRASV12.

Benralizumab treatment in an elderly patient with eosinophilic esophagitis resulted in remission: a case report.

BACKGROUND: Interleukin-5 (IL-5) has recently been shown to play a crucial role in eosinophil-mediated diseases, implying that an IL-5 receptor alpha chain (IL-5Rα) antibody (benralizumab) can be effective against eosinophilic esophagitis (EoE). Here, we present a case in which benralizumab significantly improved the symptoms and signs of an elderly Asian woman with EoE who had inadequate response to existing treatments. Case presentation A 73-year-old woman with an 8-year history of bronchial asthma (BA) and a 7-year history of dysphagia presented to our hospital with worsening dysphagia, vomiting, chest pain, and difficulty in eating. Blood biochemical findings revealed an increase in the eosinophil fraction of white blood cells (42.2%), and a conventional chest computed tomography scan revealed esophageal wall thickening. An upper gastrointestinal endoscopy revealed mucosal edema as well as multiple esophageal rings, and esophageal biopsy specimens showed an eosinophilic infiltrate of more than 15 cells/ high power field. Based on these findings, she was diagnosed as EoE complicated by BA. We firstly administrated 20 mg/day of prednisolone, rabeprazole sodium and liquid budesonide oral suspension for 5 months; however, they were ineffective and her dysphagia worsened over time. Then, benralizumab treatment in combination with these drugs was started. Her dysphagia completely disappeared 2 weeks after starting benralizumab, and an upper endoscopy showed that the clinical findings had completely disappeared after another 6 weeks. Benralizumab was then given to her for 41 months, and her symptoms remained in remission. In addition, she had no EoE recurrence for more than 12 months after discontinuing benralizumab. CONCLUSIONS: Benralizumab in combination with other multiple drugs significantly improved the symptoms and examination findings of an elderly patients with EoE. Furthermore, she experienced no recurrence even after discontinuing benralizumab withdrawal, suggesting that benralizumab could be an appropriate therapeutic option for EoE.

Circulating microRNA Panel for Prediction of Recurrence and Survival in Early-Stage Lung Adenocarcinoma.

Early-stage lung adenocarcinoma (LUAD) patients remain at substantial risk for recurrence and disease-related death, highlighting the unmet need of biomarkers for the assessment and identification of those in an early stage who would likely benefit from adjuvant chemotherapy. To identify circulating miRNAs useful for predicting recurrence in early-stage LUAD, we performed miRNA microarray analysis with pools of pretreatment plasma samples from patients with stage I LUAD who developed recurrence or remained recurrence-free during the follow-up period. Subsequent validation in 85 patients with stage I LUAD resulted in the development of a circulating miRNA panel comprising miR-23a-3p, miR-320c, and miR-125b-5p and yielding an area under the curve (AUC) of 0.776 in predicting recurrence. Furthermore, the three-miRNA panel yielded an AUC of 0.804, with a sensitivity of 45.8% at 95% specificity in the independent test set of 57 stage I and II LUAD patients. The miRNA panel score was a significant and independent factor for predicting disease-free survival (p < 0.001, hazard ratio [HR] = 1.64, 95% confidence interval [CI] = 1.51-4.22) and overall survival (p = 0.001, HR = 1.51, 95% CI = 1.17-1.94). This circulating miRNA panel is a useful noninvasive tool to stratify early-stage LUAD patients and determine an appropriate treatment plan with maximal efficacy.

Inhalation adherence for asthma and COPD improved during the COVID-19 pandemic: a questionnaire survey at a university hospital in Japan.

Background: Good adherence to an inhaled medication protocol is necessary for the management of asthma and chronic obstructive pulmonary disease (COPD), and several interventions to improve adherence have been reported. However, the impact of patient life changes and psychological aspects on treatment motivation is obscure. Here, we investigated changes in inhaler adherence during the COVID-19 pandemic and how lifestyle and psychological changes affected it.Methods: Seven-hundred sixteen adult patients with asthma and COPD who had visited Nagoya University Hospital between 2015 and 2020 were selected. Among them, 311 patients had received instruction at a pharmacist-managed clinic (PMC). We distributed one-time cross-sectional questionnaires from January 12 to March 31, 2021. The questionnaire covered the status of hospital visits, inhalation adherence before and during the COVID-19 pandemic, lifestyles, medical conditions, and psychological stress. The Adherence Starts with Knowledge-12 (ASK-12) was used to assess adherence barriers.Results: Four-hundred thirty-three patients answered the questionnaire. Inhalation adherence was significantly improved in both diseases during the COVID-19 pandemic. The most common reason for improved adherence was fear of infection. Patients with improved adherence were more likely to believe that controller inhalers could prevent COVID-19 from becoming more severe. Improved adherence was more common in patients with asthma, those not receiving counseling at PMC, and those with poor baseline adherence.Conclusions: Inhalation adherence for asthma and COPD improved in the COVID-19 pandemic. The patients seemed to realize the necessity and benefits of the medication more strongly than before the pandemic, which motivated them to improve adherence.

Resistance to mutant KRASV12-induced senescence in an hTERT/Cdk4-immortalized normal human bronchial epithelial cell line.

Mutant KRAS, the most frequently occurring (∼30%) driver oncogene in lung adenocarcinoma, induces normal epithelial cells to undergo senescence. This phenomenon, called "oncogene-induced senescence (OIS)", prevents mutant KRAS-induced malignant transformation. We have previously reported that mutant KRASV12 induces OIS in a subset of normal human bronchial epithelial cell line immortalized with hTERT and Cdk4. Understanding the mechanism and efficacy of this important cancer prevention mechanism is a key knowledge gap. Therefore, this study investigates mutant KRASV12-induced OIS in upregulated telomerase combined with the p16/RB pathway inactivation in normal bronchial epithelial cells. The normal (non-transformed and non-tumorigenic) human bronchial epithelial cell line HBEC3 (also called "HBEC3KT"), immortalized with hTERT ("T") and Cdk4 ("K"), was used in this study. HBEC3 that expressed mutant KRASV12 in a doxycycline-regulated manner was established (designated as HBEC3-RIN2). Controlled induction of mutant KRASV12 expression induced partial epithelial-to-mesenchymal transition in HBEC3-RIN2 cells, which was associated with upregulated expression of ZEB1 and SNAIL. Mutant KRASV12 caused the majority of HBEC3-RIN2 to undergo morphological changes; suggestive of senescence, which was associated with enhanced autophagic flux. Upon mutant KRASV12 expression, only a small HBEC3-RIN2 cell subset underwent senescence, as assessed by a senescence-associated ß-galactosidase staining (SA-ßG) method. Furthermore, mutant KRASV12 enhanced cell growth, evaluated by colorimetric proliferation assay, and liquid and soft agar colony formation assays, partially through increased phosphorylated AKT and ERK expression but did not affect cell division, or cell cycle status. Intriguingly, mutant KRASV12 reduced p53 protein expression but increased p21 protein expression by prolonging its half-life. These results indicate that an hTERT/Cdk4 -immortalized normal bronchial epithelial cell line is partially resistant to mutant KRASV12-induced senescence. This suggests that OIS does not efficiently suppress KRASV12-induced transformation in the context of the simultaneous occurrence of telomerase upregulation and inactivation of the p16/Rb pathway.

Clinical efficacy of osimertinib in EGFR-mutant non-small cell lung cancer with distant metastasis.

BACKGROUND: Osimertinib-the third-generation epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI)-has been widely used as a first-line treatment for patients with metastatic EGFR-mutant non-small cell lung cancer (NSCLC). Osimertinib demonstrated central nervous system activity in patients with brain metastasis; however, its efficacy against other distant metastatic organs, including bone and liver, remains unclear. Therefore, we retrospectively analyzed the clinical efficacy of osimertinib in these patients in comparison to other EGFR-TKIs. METHODS: Clinical data of patients with advanced NSCLC receiving gefitinib/erlotinib (n = 183), afatinib (n = 55), or osimertinib (n = 150) at five medical institutions were retrospectively assessed for progression-free survival (PFS), overall survival (OS), and best overall response rate (ORR). RESULTS: In univariate and multivariate analyses, most distant metastases, including the brain and bone, were unrelated to the therapeutic efficacy of osimertinib, although liver metastasis and L858R mutation were independently associated with shorter PFS. PFS and OS in patients with liver metastases were significantly shorter than those in patients without liver metastases (PFS: 7.4 vs. 19.7 months, OS: 12.1 months vs. not reached, respectively). Osimertinib provided significantly longer PFS in patients with brain or bone metastasis and exon 19 deletion than the other EGFR-TKIs. The PFS of patients with liver metastases was not significantly different among the three EGFR-TKI groups. Furthermore, the ORR of osimertinib in patients with liver metastases was significantly attenuated, and the effectiveness was similar to 1st- or 2nd -generation EGFR-TKIs. CONCLUSION: Osimertinib provided better clinical benefits than 1st- and 2nd-generation EGFR-TKIs for patients with EGFR-mutant NSCLC, particularly those with brain or bone metastases and exon 19 deletion; however, its efficacy against liver metastasis was remarkably attenuated. New therapeutic developments for patients with EGFR-mutant NSCLC with liver metastases are needed.

Oxytocin receptor is a promising therapeutic target of malignant mesothelioma.

Malignant mesothelioma (MM) is one of the most aggressive tumors. We conducted bioinformatics analysis using Cancer Cell Line Encyclopedia (CCLE) datasets to identify new molecular markers in MM. Overexpression of oxytocin receptor (OXTR), which is a G-protein-coupled receptor for the hormone and neurotransmitter oxytocin, mRNA was distinctively identified in MM cell lines. Therefore, we assessed the role of OXTR and its clinical relevance in MM. Kaplan-Meier and Cox regression analyses were applied to assess the association between overall survival and OXTR mRNA expression using The Cancer Genome Atlas (TCGA) datasets. The function of OXTR and the efficacy of its antagonists were investigated in vitro and in vivo using MM cell lines. Consistent with the findings from CCLE datasets analysis, OXTR mRNA expression was highly increased in MM tissues compared with other cancer types in the TCGA datasets, and MM cases with high OXTR expression showed poor overall survival. Moreover, OXTR knockdown dramatically decreased MM cell proliferation in cells with high OXTR expression via tumor cell cycle disturbance, whereas oxytocin treatment significantly increased MM cell growth. OXTR antagonists, which have high selectivity for OXTR, inhibited the growth of MM cell lines with high OXTR expression, and oral administration of the OXTR antagonist, cligosiban, significantly suppressed MM tumor progression in a xenograft model. Our findings suggest that OXTR plays a crucial role in MM cell proliferation and is a promising therapeutic target that may broaden potential therapeutic options and could be a prognostic biomarker of MM.

The current issues and future perspective of artificial intelligence for developing new treatment strategy in non-small cell lung cancer: harmonization of molecular cancer biology and artificial intelligence.

Comprehensive analysis of omics data, such as genome, transcriptome, proteome, metabolome, and interactome, is a crucial technique for elucidating the complex mechanism of cancer onset and progression. Recently, a variety of new findings have been reported based on multi-omics analysis in combination with various clinical information. However, integrated analysis of multi-omics data is extremely labor intensive, making the development of new analysis technology indispensable. Artificial intelligence (AI), which has been under development in recent years, is quickly becoming an effective approach to reduce the labor involved in analyzing large amounts of complex data and to obtain valuable information that is often overlooked in manual analysis and experiments. The use of AI, such as machine learning approaches and deep learning systems, allows for the efficient analysis of massive omics data combined with accurate clinical information and can lead to comprehensive predictive models that will be desirable for further developing individual treatment strategies of immunotherapy and molecular target therapy. Here, we aim to review the potential of AI in the integrated analysis of omics data and clinical information with a special focus on recent advances in the discovery of new biomarkers and the future direction of personalized medicine in non-small lung cancer.

Efficacies of programmed cell death 1 ligand 1 blockade in non-small cell lung cancer patients with acquired resistance to prior programmed cell death 1 inhibitor and development of diabetic ketoacidosis caused by two different etiologies: a retrospective case series.

The programmed cell death 1 (PD-1)/programmed cell death 1 ligand 1 (PD-L1) axis is vital for immune resistance during tumor development, while PD-L1 inhibitors can also inhibit the PD-L1/B7-1 (CD80) interaction, indicating one of the molecular differences between PD-1 and PD-L1 inhibitors. However, the clinical benefits of PD-L1 inhibitors in patients previously treated with PD-1 inhibitors remain unknown. In this study, we retrospectively analyzed the clinical data of eight patients with non-small cell lung cancer who received the PD-L1 inhibitor atezolizumab and previously treated with the PD-1 inhibitor nivolumab. The median progression-free survival was 2.1 months (1.8-18.7 months), and 4 of 8 patients achieved at least stable disease. In two of these patients, atezolizumab treatment resulted in longer progression-free survival (PFS) compared with that of nivolumab. Conversely, one patient exhibited grade 4 diabetic ketoacidosis (DKA) within 2 weeks after the initial administration of atezolizumab. Another patient had developed type 1 diabetes mellitus (T1DM) during the prior nivolumab treatment and then developed DKA due to an infection after the initiation of atezolizumab. Both of them had high-risk human leukocyte antigen-DR/DQ types relevant to T1DM. These results demonstrate the potential efficacy of PD-L1 inhibitors to some tumors that have acquired resistance to PD-1 inhibitors and suggest that appropriate managements are required for not only a newly onset of T1DM but also blood glucose control after the development of T1DM during a reiteration of the PD-1/PD-L1 blockade.

Current Immunotherapeutic Strategies Targeting the PD-1/PD-L1 Axis in Non-Small Cell Lung Cancer with Oncogenic Driver Mutations.

Treatment strategies targeting programed cell death 1 (PD-1) or its ligand, PD-L1, have been developed as immunotherapy against tumor progression for various cancer types including non-small cell lung cancer (NSCLC). The recent pivotal clinical trials of immune-checkpoint inhibiters (ICIs) combined with cytotoxic chemotherapy have reshaped therapeutic strategies and established various first-line standard treatments. The therapeutic effects of ICIs in these clinical trials were analyzed according to PD-L1 tumor proportion scores or tumor mutational burden; however, these indicators are insufficient to predict the clinical outcome. Consequently, molecular biological approaches, including multi-omics analyses, have addressed other mechanisms of cancer immune escape and have revealed an association of NSCLC containing specific driver mutations with distinct immune phenotypes. NSCLC has been characterized by driver mutation-defined molecular subsets and the effect of driver mutations on the regulatory mechanism of PD-L1 expression on the tumor itself. In this review, we summarize the results of recent clinical trials of ICIs in advanced NSCLC and the association between driver alterations and distinct immune phenotypes. We further discuss the current clinical issues with a future perspective for the role of precision medicine in NSCLC.

Primary Prophylaxis Indication for Docetaxel Induced Febrile Neutropenia in Elderly Patients with Non-Small Cell Lung Cancer.

In advanced non-small cell lung cancer (NSCLC), the reported incidence of febrile neutropenia (FN) caused by docetaxel (DTX) is 10-20% in clinical trial data. However, FN incidence caused by DTX in real-world setting remains unclear. We evaluated FN incidence caused by DTX and identify risk factors of FN in real-world setting. One hundred and seventy-one NSCLC patients treated with DTX were retrospectively analyzed and 44 (26%) developed FN. Multivariate analysis identified higher age (≥65 years) and prior history of FN as independent risk factors for FN. Primary prophylaxis for FN might be recommended in elderly patients with/without prior history of FN.

UHRF1, a Regulator of Methylation, as a Diagnostic and Prognostic Marker for Lung Cancer.

We evaluated the value of UHRF1, a regulator of methylation, as a biomarker for lung cancer. UHRF1 is expressed at higher levels in both lung adenocarcinoma (AD) and squamous cell carcinoma (SQ); however, a meta-analysis showed that UHRF1 expression is correlated with worse survival in patients with AD but not in those with SQ. UHRF1 knockdown suppressed the growth of lung cancer cell lines through G1 cell cycle arrest in some cell lines. These results suggest that UHRF1 may server as a diagnostic marker for AD and SQ and as a prognostic marker for AD in lung cancer.

CADM1 associates with Hippo pathway core kinases; membranous co-expression of CADM1 and LATS2 in lung tumors predicts good prognosis.

Cell adhesion molecule-1 (CADM1) is a member of the immunoglobulin superfamily that functions as a tumor suppressor of lung tumors. We herein demonstrated that CADM1 interacts with Hippo pathway core kinases and enhances the phosphorylation of YAP1, and also that the membranous co-expression of CADM1 and LATS2 predicts a favorable prognosis in lung adenocarcinoma. CADM1 significantly repressed the saturation density elevated by YAP1 overexpression in NIH3T3 cells. CADM1 significantly promoted YAP1 phosphorylation on Ser 127 and downregulated YAP1 target gene expression at confluency in lung adenocarcinoma cell lines. Moreover, CADM1 was co-precipitated with multiple Hippo pathway components, including the core kinases MST1/2 and LATS1/2, suggesting the involvement of CADM1 in the regulation of the Hippo pathway through cell-cell contact. An immunohistochemical analysis of primary lung adenocarcinomas (n = 145) revealed that the histologically low-grade subtype frequently showed the membranous co-expression of CADM1 (20/22, 91% of low-grade; 61/91, 67% of intermediate grade; and 13/32, 41% of high-grade subtypes; P < 0.0001) and LATS2 (22/22, 100% of low-grade; 44/91, 48% of intermediate-grade; and 1/32, 3% of high-grade subtypes; P < 0.0001). A subset analysis of disease-free survival revealed that the membranous co-expression of CADM1 and LATS2 was a favorable prognosis factor (5-year disease-free survival rate: 83.8%), even with nuclear YAP1-positive expression (5-year disease-free survival rate: 83.7%), whereas nuclear YAP1-positive cases with the negative expression of CADM1 and LATS2 had a poorer prognosis (5-year disease-free survival rate: 33.3%). These results indicate that the relationship between CADM1 and Hippo pathway core kinases at the cell membrane is important for suppressing the oncogenic role of YAP1.

eIF2ß, a subunit of translation-initiation factor EIF2, is a potential therapeutic target for non-small cell lung cancer.

To identify novel therapeutic targets for non-small cell lung cancer (NSCLC), we conducted an integrative study in the following 3 stages: (i) identification of potential target gene(s) through shRNA functional screens in 2 independent NSCLC cell lines; (ii) validation of the clinical relevance of identified gene(s) using public databases; and (iii) investigation of therapeutic potential of targeting the identified gene(s) in vitro. A semi-genome-wide shRNA screen was performed in NCI-H358 cells, and was integrated with data from our previous screen in NCI-H460 cells. Among genes identified in shRNA screens, 24 were present in both NCI-H358 and NCI-H460 cells and were considered potential targets. Among the genes, we focused on eIF2ß, which is a subunit of heterotrimeric G protein EIF2 and functions as a transcription initiation factor. The eIF2ß protein is highly expressed in lung cancer cell lines compared with normal bronchial epithelial cells, and gene copy number analyses revealed that eIF2ß is amplified in a subset of NSCLC cell lines. Gene expression analysis using The Cancer Genome Atlas (TCGA) dataset revealed that eIF2ß expression is significantly upregulated in lung cancer tissues compared with corresponding normal lung tissues. Furthermore, high eIF2ß expression was correlated with poor survival in patients with lung adenocarcinoma, as shown in other cohorts using publicly available online tools. RNAi-mediated depletion of eIF2ß suppresses growth of lung cancer cells independently of p53 mutation status, in part through G1 cell cycle arrest. Our data suggest that eIF2ß is a therapeutic target for lung cancer.

Regulation of PD-L1 expression by matrix stiffness in lung cancer cells.

Expression of programmed death-ligand 1 (PD-L1) in tumor cells such as lung cancer cells plays an important role in mechanisms underlying evasion of an immune check point system. Lung cancer tissue with increased deposition of extracellular matrix is much stiffer than normal lung tissue. There is emerging evidence that the matrix stiffness of cancer tissue affects the phenotypes and properties of cancer cells. Nevertheless, the effects of substrate rigidity on expression of PD-L1 in lung cancer cells remain elusive. We evaluated the effects of substrate stiffness on PD-L1 expression in HCC827 lung adenocarcinoma cells by using polyacrylamide hydrogels with stiffnesses of 2 and 25 kPa. Expression of PD-L1 protein was higher on the stiffer substrates (25 kPa gel and plastic dish) than on the soft 2 kPa gel. PD-L1 expression was reduced by detachment of cells adhering to the substrate. Interferon-γ enhanced expression of PD-L1 protein cultured on stiff (25 kPa gel and plastic dishes) and soft (2 kPa gel) substrates and in the cell adhesion-free condition. As the stiffness of substrates increased, formation of actin stress fiber and cell growth were enhanced. Transfection of the cells with short interfering RNA for PD-L1 inhibited cell growth without affecting stress fiber formation. Treatment of the cells with cytochalasin D, an inhibitor of actin polymerization, significantly reduced PD-L1 protein levels. Taken together, a stiff substrate enhanced PD-L1 expression via actin-dependent mechanisms in lung cancer cells. It is suggested that stiffness as a tumor environment regulates PD-L1 expression, which leads to evasion of the immune system and tumor growth.

Predictive and Prognostic Molecular Biomarkers for Response to Neoadjuvant Chemoradiation in Rectal Cancer.

The standard of care in locally advanced rectal cancer is neoadjuvant chemoradiation (nCRT) followed by radical surgery. Response to nCRT varies among patients and pathological complete response is associated with better outcome. However, there is a lack of effective methods to select rectal cancer patients who would or would not have a benefit from nCRT. The utility of clinicopathological and radiological features are limited due to lack of adequate sensitivity and specificity. Molecular biomarkers have the potential to predict response to nCRT at an early time point, but none have currently reached the clinic. Integration of diverse types of biomarkers including clinicopathological and imaging features, identification of mechanistic link to tumor biology, and rigorous validation using samples which represent disease heterogeneity, will allow to develop a sensitive and cost-effective molecular biomarker panel for precision medicine in rectal cancer. Here, we aim to review the recent advance in tissue- and blood-based molecular biomarker research and illustrate their potential in predicting nCRT response in rectal cancer.

Loss of YAP1 defines neuroendocrine differentiation of lung tumors.

YAP1, the main Hippo pathway effector, is a potent oncogene and is overexpressed in non-small-cell lung cancer (NSCLC); however, the YAP1 expression pattern in small-cell lung cancer (SCLC) has not yet been elucidated in detail. We report that the loss of YAP1 is a special feature of high-grade neuroendocrine lung tumors. A hierarchical cluster analysis of 15 high-grade neuroendocrine tumor cell lines containing 14 SCLC cell lines that depended on the genes of Hippo pathway molecules and neuroendocrine markers clearly classified these lines into two groups: the YAP1-negative and neuroendocrine marker-positive group (n = 11), and the YAP1-positive and neuroendocrine marker-negative group (n = 4). Among the 41 NSCLC cell lines examined, the loss of YAP1 was only observed in one cell line showing the strong expression of neuroendocrine markers. Immunostaining for YAP1, using the sections of 189 NSCLC, 41 SCLC, and 30 large cell neuroendocrine carcinoma (LCNEC) cases, revealed that the loss of YAP1 was common in SCLC (40/41, 98%) and LCNEC (18/30, 60%), but was rare in NSCLC (6/189, 3%). Among the SCLC and LCNEC cases tested, the loss of YAP1 correlated with the expression of neuroendocrine markers, and a survival analysis revealed that YAP1-negative cases were more chemosensitive than YAP1-positive cases. Chemosensitivity test for cisplatin using YAP1-positive/YAP1-negative SCLC cell lines also showed compatible results. YAP1-sh-mediated knockdown induced the neuroendocrine marker RAB3a, which suggested the possible involvement of YAP1 in the regulation of neuroendocrine differentiation. Thus, we showed that the loss of YAP1 has potential as a clinical marker for predicting neuroendocrine features and chemosensitivity.

The oncogenic role of LGR6 overexpression induced by aberrant Wnt/ß-catenin signaling in lung cancer.

BACKGROUND: Molecular abnormalities in the Wnt/ß-catenin pathway confer malignant phenotypes in lung cancer. Previously, we identified the association of leucine-rich repeat-containing G protein-coupled receptor 6 (LGR6) with oncogenic Wnt signaling, and its downregulation upon ß-catenin knockdown in non-small cell lung cancer (NSCLC) cells carrying CTNNB1 mutations. The aim of this study was to explore the mechanisms underlying this association and the accompanying phenotypes. METHODS: LGR6 expression in lung cancer cell lines and surgical specimens was analyzed using quantitative RT-PCR and immunohistochemistry. Cell growth was assessed using colony formation assay. Additionally, mRNA sequencing was performed to compare the expression profiles of cells subjected to different treatments. RESULTS: LGR6 was overexpressed in small cell lung cancer (SCLC) and NSCLC cell lines, including the CTNNB1-mutated NSCLC cell lines HCC15 and A427. In both cell lines, LGR6 knockdown inhibited cell growth. LGR6 expression was upregulated in spheroids compared to adherent cultures of A427 cells, suggesting that LGR6 participates in the acquisition of cancer stem cell properties. Immunohistochemical analysis of lung cancer specimens revealed that the LGR6 protein was predominantly overexpressed in SCLCs, large cell neuroendocrine carcinomas, and lung adenocarcinomas, wherein LGR6 overexpression was associated with vascular invasion, the wild-type EGFR genotype, and an unfavorable prognosis. Integrated mRNA sequencing analysis of HCC15 and A427 cells with or without LGR6 knockdown revealed LGR6-related pathways and genes associated with cancer development and stemness properties. CONCLUSIONS: Our findings highlight the oncogenic roles of LGR6 overexpression induced by aberrant Wnt/ß-catenin signaling in lung cancer.

Candidate tumor-specific CD8+ T cell subsets identified in the malignant pleural effusion of advanced lung cancer patients by single-cell analysis.

Isolation of tumor-specific T cells and their antigen receptors (TCRs) from malignant pleural effusions (MPE) may facilitate the development of TCR-transduced adoptive cellular immunotherapy products for advanced lung cancer patients. However, the characteristics and markers of tumor-specific T-cells in MPE are largely undefined. To this end, to establish the phenotypes and antigen specificities of CD8+ T cells, we performed single-cell RNA and TCR sequencing of samples from three advanced lung cancer patients. Dimensionality reduction on a total of 4,983 CD8+ T cells revealed 10 clusters including naïve, memory, and exhausted phenotypes. We focused particularly on exhausted T cell clusters and tested their TCR reactivity against neoantigens predicted from autologous cancer cell lines. Four different TCRs specific for the same neoantigen and one orphan TCR specific for the autologous cell line were identified from one of the patients. Differential gene expression analysis in tumor-specific T cells relative to the other T cells identified CXCL13, as a candidate gene expressed by tumor-specific T cells. In addition to expressing CXCL13, tumor-specific T cells were present in a higher proportion of T cells co-expressing PDCD1(PD-1)/TNFRSF9(4-1BB). Furthermore, flow cytometric analyses in advanced lung cancer patients with MPE documented that those with high PD-1/4-1BB expression have a better prognosis in the subset of 57 adenocarcinoma patients (p = .039). These data suggest that PD-1/4-1BB co-expression might identify tumor-specific CD8+ T cells in MPE, which are associated with patients' prognosis. (233 words).

Metabolic barriers in non-small cell lung cancer with LKB1 and/or KEAP1 mutations for immunotherapeutic strategies.

Currently, immune checkpoint inhibitors (ICIs) are widely considered the standard initial treatment for advanced non-small cell lung cancer (NSCLC) when there are no targetable driver oncogenic alternations. NSCLC tumors that have two alterations in tumor suppressor genes, such as liver kinase B1 (LKB1) and/or Kelch-like ECH-associated protein 1 (KEAP1), have been found to exhibit reduced responsiveness to these therapeutic strategies, as revealed by multiomics analyses identifying immunosuppressed phenotypes. Recent advancements in various biological approaches have gradually unveiled the molecular mechanisms underlying intrinsic reprogrammed metabolism in tumor cells, which contribute to the evasion of immune responses by the tumor. Notably, metabolic alterations in glycolysis and glutaminolysis have a significant impact on tumor aggressiveness and the remodeling of the tumor microenvironment. Since glucose and glutamine are essential for the proliferation and activation of effector T cells, heightened consumption of these nutrients by tumor cells results in immunosuppression and resistance to ICI therapies. This review provides a comprehensive summary of the clinical efficacies of current therapeutic strategies against NSCLC harboring LKB1 and/or KEAP1 mutations, along with the metabolic alterations in glycolysis and glutaminolysis observed in these cancer cells. Furthermore, ongoing trials targeting these metabolic alterations are discussed as potential approaches to overcome the extremely poor prognosis associated with this type of cancer.