Anti-Müllerian hormone type II receptor protein expression in non-small cell lung cancer and the effect of AMH/AMHR2 signaling on cancer cell proliferation.

BACKGROUND: Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths worldwide despite advances in cancer therapeutics. In several gynecological cancers, anti-Müllerian hormone receptor type 2 (AMHR2) mediates AMH-induced growth inhibition and is expressed at high levels. Furthermore, 5%-8% of NSCLCs exhibit high AMHR2 expression, suggesting that AMH may inhibit the progression of some lung cancers. However, the clinical relevance of AMHR2 expression and its role in lung cancer is not fully clarified. METHODS: Immunostaining was performed on 79 surgical specimens of NSCLC. The Cancer Genome Atlas RNA-seq data for lung adenocarcinoma were analyzed, and gene ontology and gene set enrichment analyses were performed. In cellular experiments, AMHR2-overexpressing NSCLC cell lines were established, and the role of the AMH-AMHR2 pathway in cell proliferation with recombinant human AMH protein treatment was examined. RESULTS: A total of 13 cases (16.5%) were positive for immunostaining in lung adenocarcinoma tissues; no positive signals were detected in lung squamous carcinoma tissues. Gene expression variation analysis using The Cancer Genome Atlas data showed that the expression of genes related to the cell cycle was downregulated in the AMHR2-high group. Cellular experiments showed that activation of the AMH-AMHR2 pathway suppressed cell proliferation. CONCLUSION: In lung adenocarcinoma tissues with high expression of AMHR2, activation of the AMH-AMHR2 pathway may suppress cell proliferation.

Clinical potential of SKP2 as diagnostic marker and therapeutic target in small cell lung cancer.

BACKGROUND: Small cell lung cancer (SCLC) is the most aggressive type of lung cancer. The overall survival has not improved significantly over the last decades because no major therapeutic breakthroughs have been achieved for over 15 years. METHODS: We analyzed a genome-wide loss-of-function screening database to identify vulnerabilities in SCLC for the development of urgently needed novel therapies. RESULTS: We identified SKP2 (encoding S-phase kinase-associated protein 2) and CKS1B (encoding CDC28 protein kinase regulatory subunit 1B) as the two most essential genes in that order in SCLC. Notably, SKP2 and CKS1B comprise the p27 binding pocket of the E3 ubiquitin ligase SCFSKP2 complex. Immunohistochemistry on tissue microarrays revealed that SKP2 was expressed in >95% of samples at substantially higher levels than that observed for commonly used neuroendocrine markers. As expected, SCLC cell lines were sensitive to SKP2 inhibition. Furthermore, SKP2 or CKS1B knockdown induced apoptosis in RB1 mutant cells, whereas it induced senescence in RB1 wild-type cells. CONCLUSION: Although the mechanism underlying SKP2 knockdown-induced growth inhibition differs between RB1-wild-type and -mutant SCLC, SKP2 can be considered a novel therapeutic target for patients with SCLC regardless of the RB1 mutation status. Our findings indicate that SKP2 is a potential novel clinical diagnostic marker and therapeutic target in SCLC.

CALML5 is a novel diagnostic marker for differentiating thymic squamous cell carcinoma from type B3 thymoma.

BACKGROUND: Thymic squamous cell carcinoma and type B3 thymoma are primary neoplasms of the anterior mediastinum that are sometimes difficult to differentiate from one another histologically. However, only a few immunohistochemical markers are available for the differential diagnosis. The purpose of this study was to discover a novel marker for differentiating between thymic squamous cell carcinoma and type B3 thymoma. METHODS: We used histological samples of thymic carcinomas (n = 26) and type B3 thymomas (n = 38) which were resected between 1986 and 2017. To search for candidates of differential markers, gene expression levels were evaluated in samples using promoter analysis by cap analysis of gene expression (CAGE) sequencing. RESULTS: Promoter level expression of CALML5 genes was significantly higher in thymic carcinomas than in type B3 thymomas. We further validated the results of the CAGE analysis in all 26 thymic carcinomas and 38 type B3 thymomas by immunohistochemistry (IHC). CALML5 was strongly expressed in the cytoplasm in 19 of 26 cases with thymic carcinoma, whereas positivity at the protein level was shown in two of 38 type B3 thymomas. Thus, the sensitivity (73.1%) and specificity (94.7%) of CALML5 as markers for immunohistochemical diagnosis of thymic carcinoma were extremely high. CONCLUSION: We identified CALML5 as a potential marker for differentiating thymic squamous cell carcinoma from type B3 thymoma. It is assumed that future clinical use of CALML5 may improve the diagnostic accuracy of differentiating between these two diseases.

Early Detection of Therapeutic Benefit from PD-1/PD-L1 Blockade in Advanced Lung Cancer by Monitoring Cachexia-Related Circulating Cytokines.

Cancer cachexia is associated with poor immunotherapeutic outcomes. This prospective observational study longitudinally evaluated the role of cachexia-related circulating cytokines in predicting the risk and benefit of PD-1/PD-L1 blockade in advanced lung cancer. Forty-one circulating cytokines at baseline and after one cycle of PD-1/PD-L1 blockade treatment were measured in patients with advanced lung cancer between 2019 and 2020. The cachexia-related cytokines were identified by comparing the levels of circulating cytokines between cachectic and non-cachectic patients. Among 55 patients, 49.1% were diagnosed with cachexia at the beginning of PD-1/PD-L1 blockade therapy. Baseline levels of the circulating cytokines IL-6, IL-8, IL-10, IL-15, and IP-10 were significantly higher in cachectic patients. In contrast, the level of eotaxin-1 was lower in cachectic patients than in those without cachexia. Higher IL-6 at baseline and during treatment was associated with a greater risk of immune-related adverse events, while higher IL-10 at baseline was linked to worse overall survival. More importantly, increased eotaxin-1 after one cycle of PD-1/PD-L1 blockade treatment was associated with higher objective response and better overall survival. A blood-based, cachexia-related cytokine assay may yield potential biomarkers for the early prediction of clinical response to PD-1/PD-L1 blockade and provide clues for improving the outcomes of cachectic patients.

A Novel Therapeutic Strategy Targeting the Mesenchymal Phenotype of Malignant Pleural Mesothelioma by Suppressing LSD1.

Malignant pleural mesothelioma (MPM) is a highly aggressive tumor that has a low overall survival; however, no significant treatment advances have been made in the past 15 years. Large-scale molecular studies have identified a poor prognostic subset of MPM linked to the epithelial-mesenchymal transition (EMT) that may contribute toward resistance to chemotherapy, suggesting that EMT could be targeted to treat patients with MPM. Previously, we reported that histone modifiers regulating EMT could be therapeutic targets; therefore, in this study, we investigated whether targeting lysine-specific demethylase 1 (LSD1/KDM1), a histone-modifying enzyme responsible for demethylating histone H3 lysine 4 and lysine 9, could represent a novel therapeutic strategy for MPM. We suppressed LSD1 and investigated the EMT phenotype using EMT marker expression and wound-healing assay; and chemosensitivity using apoptosis assay. We found that suppressing LSD1 induces an epithelial phenotype in sarcomatoid MPM cells, while attenuating the mesenchymal phenotype sensitized MPM cells to cisplatin-induced apoptosis. Subsequent genome-wide identification, comprehensive microarray analysis, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) to assess genome-wide changes in chromatin accessibility suggested that LSD1 directly regulates milk fat globulin protein E8 (MFGE8), an integrin ligand that is involved in the FAK pathway. Furthermore, we found that LSD1 regulates the mesenchymal phenotype and apoptosis by activating the FAK-AKT-GSK3ß pathway via a positive feedback loop involving MFGE8 and Snail expression, thereby leading to cisplatin resistance. IMPLICATIONS: This study suggests that LSD1 regulates the mesenchymal phenotype and apoptosis, and that LSD1 inhibitors could be combined with the cisplatin as a novel therapy for patients with MPM.

Zinc-finger E-box-binding homeobox 1 (ZEB1) plays a crucial role in the maintenance of lung cancer stem cells resistant to gefitinib.

BACKGROUND: Zinc-finger E-box-binding homeobox 1 (ZEB1) is an important regulator of epithelial-mesenchymal transition (EMT) and is involved in the maintenance of cancer stem cells (CSCs) via miR-200c and BMI1 pathway. Recent studies revealed that ZEB1 contributes to the EMT-mediated acquired resistance to gefitinib in EGFR-mutant non-small cell lung cancer (NSCLC). However, the precise role of ZEB1 in the maintenance of lung CSCs that lead to acquired resistance to gefitinib remains unclear. METHODS: PC9 and HCC827 NSCLC cell lines were treated with high concentrations of gefitinib, and surviving cells were referred to as "gefitinib-resistant persisters" (GRPs). ZEB1 knockdown or overexpression was performed to determine the biological significance of ZEB1 in the CSC features of GRPs, and animal models were studied for in vivo validation. Expression of ZEB1, BMI1, and ALDH1A1 was analyzed by immunohistochemistry in tumor specimens from NSCLC patients with acquired resistance to gefitinib. RESULTS: GRPs had characteristic features of mesenchymal and CSC phenotypes with high expression of ZEB1 and BMI1, and decreased miR-200c, in vitro and in vivo. ZEB1 silencing attenuated the suppression of miR-200c, resulting in the reduction in BMI1 and reversed the mesenchymal and CSC features of GRPs. Furthermore, ZEB1 overexpression induced EMT and increased the levels of CD133- and BMI1-positive GRPs in vitro and gefitinib resistance in vivo. Finally, ZEB1, BMI1, and ALDH1A1 were highly expressed in tumor specimens from EGFR-mutant NSCLC patients with gefitinib resistance. CONCLUSIONS: ZEB1 plays an important role in gefitinib-resistant lung CSCs with EMT features via regulation of miR-200c and BMI1.

The use of indoor plant as an alternative strategy to improve indoor air quality in Indonesia.

Indoor air pollution marked with decreased air quality below the set standard. The quality of indoor air is determined by ambient air quality as well as by a harmful substance resulting from the household activity. Indoor air pollution may cause several problems such as sick building syndrome, chronic obstructive pulmonary disease (COPD), asthma, lung cancer, and is responsible for nearly two million death in developing countries. One of the interesting research topics to overcome the indoor air pollution problem is the application of indoor plants. Although there are no established criteria to specify the best indoor plant, several studies have revealed the capability of a particular indoor plant to remove the harmful substances. This paper summarizes important information about indoor air pollution and provides the evidence-based insight of indoor plant usefulness as an alternative way for indoor air remediation.

Nintedanib inhibits epithelial-mesenchymal transition in A549 alveolar epithelial cells through regulation of the TGF-ß/Smad pathway.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disorder. Recent studies have suggested that epithelial-mesenchymal transition (EMT) of alveolar epithelial cells influences development of pulmonary fibrosis, which is mediated by transforming growth factor ß (TGF-ß). Tumor necrosis factor α (TNF-α), an important proinflammatory cytokine in IPF, has been shown to enhance TGF-ß-induced EMT. Nintedanib, a multiple tyrosine kinase inhibitor that is currently used to treat IPF, has been shown to suppress EMT in various cancer cell lines. However, the mechanism of EMT inhibition by nintedanib and its effect on TGF-ß and TNF-α signaling pathways in alveolar epithelial cells have not been fully elucidated. METHODS: A549 alveolar epithelial cells were stimulated with TGF-ß2 and TNF-α, and the effects of nintedanib on global gene expression were evaluated using microarray analysis. Furthermore, Smad2/3 phosphorylation was assessed using western blotting. RESULTS: We found that in A549 cells, TGF-ß2 and TNF-α treatment induces EMT, which was inhibited by nintedanib. Gene ontology analysis showed that nintedanib significantly attenuates the gene expression of EMT-related cellular pathways and the TGF-ß signaling pathway, but not in the TNF-α-mediated signaling pathway. Furthermore, hierarchical cluster analysis revealed that EMT-related genes were attenuated in nintedanib-treated cells. Additionally, nintedanib was found to markedly suppress phosphorylation of Smad2/3. CONCLUSION: Nintedanib inhibits EMT by mediating EMT-related gene expression and the TGF-ß/Smad pathway in A549 alveolar epithelial cells.

Activation of insulin-like growth factor-1 receptor confers acquired resistance to osimertinib in non-small cell lung cancer with EGFR T790M mutation.

BACKGROUND: Osimertinib (AZD9291) is a third-generation EGFR-tyrosine kinase inhibitor (TKI) that selectively inhibits the activating EGFR mutation and T790M mutation, and is currently used globally to treat EGFR-mutant non-small cell lung cancer (NSCLC). However, acquired resistance to osimertinib is inevitable. METHODS: We established osimertinib-resistant cells (PC9/T790M/AZDR and H1975/AZDR) derived from EGFR-mutant NSCLC cells harboring T790M mutation, and investigated the mechanism of acquired resistance to osimertinib by whole-exome sequencing and multiple phospho-receptor tyrosine kinase (RTK) array. A tumor specimen from an EGFR-mutant NSCLC patient with acquired resistance to osimertinib was also subjected to immunohistochemical analysis. RESULTS: Whole-exome sequencing analysis demonstrated that genetic alterations, such as acquisition of EGFR C797S, loss of T790M mutation, MET amplification, or mutated KRAS, MEK, BRAF, PIK3CA, were not detected. Analysis of phospho-RTK array revealed that insulin-like growth factor-1 receptor (IGF1R) was activated in PC9/T790M/AZDR and H1975/AZDR cells. Knockdown of IGF1R by siRNA as well as inhibition of IGF1R activation by linstinib (IGF1R inhibitor) significantly restored the sensitivity to osimertinib. Immunohistochemical analysis revealed that the expression level of phosphorylated IGF1R was higher in the tumor specimen from the EGFR-mutant NSCLC patient with acquired resistance to osimertinib than in the specimen collected prior to the treatment. CONCLUSIONS: IGF1R activation could occur following treatment with osimertinib in EGFR-mutant NSCLC with T790M mutation, and might be one of the mechanisms underlying osimertinib resistance. Combined treatment of osimertinib and IGF1R inhibitor might be effective in overcoming the acquired resistance to osimertinib induced by IGF1R activation. KEY POINTS: Significant findings of the study: Using osimertinib-resistant cells, we found that IGF1R activation induced by osimertinib treatment in EGFR-mutant NSCLC with T790M mutation is involved in resistance. Increased phosphorylation of IGF1R was observed in the tumor specimen from an EGFR-mutant NSCLC patient with acquired osimertinib resistance. WHAT THIS STUDY ADDS: IGF1R activation might be one of the mechanisms of osimertinib resistance. A combination therapy with osimertinib and an IGF1R inhibitor might be an optimal approach for overcoming the acquired resistance to osimertinib induced by IGF1R activation.

Role of FBXW7 in the quiescence of gefitinib-resistant lung cancer stem cells in EGFR-mutant non-small cell lung cancer.

Several recent studies suggest that cancer stem cells (CSCs) are involved in intrinsic resistance to cancer treatment. Maintenance of quiescence is crucial for establishing resistance of CSCs to cancer therapeutics. F-box/WD repeat-containing protein 7 (FBXW7) is a ubiquitin ligase that regulates quiescence by targeting the c-MYC protein for ubiquitination. We previously reported that gefitinib-resistant persisters (GRPs) in EGFR-mutant non-small cell lung cancer (NSCLC) cells highly expressed octamer-binding transcription factor 4 (Oct-4) as well as the lung CSC marker CD133, and they exhibited distinctive features of the CSC phenotype. However, the role of FBXW7 in lung CSCs and their resistance to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors in NSCLC is not fully understood. In this study, we developed GRPs from the two NSCLC cell lines PC9 and HCC827, which express an EGFR exon 19 deletion mutation, by treatment with a high concentration of gefitinib. The GRPs from both PC9 and HCC827 cells expressed high levels of CD133 and FBXW7, but low levels of c-MYC. Cell cycle analysis demonstrated that the majority of GRPs existed in the G0/G1 phase. Knockdown of the FBXW7 gene significantly reduced the cell number of CD133-positive GRPs and reversed the cell population in the G0/G1-phase. We also found that FBXW7 expression in CD133-positive cells was increased and c-MYC expression was decreased in gefitinib-resistant tumors of PC9 cells in mice and in 9 out of 14 tumor specimens from EGFR-mutant NSCLC patients with acquired resistance to gefitinib. These findings suggest that FBXW7 plays a pivotal role in the maintenance of quiescence in gefitinib-resistant lung CSCs in EGFR mutation-positive NSCLC.

Host genome polymorphisms and tuberculosis infection: What we have to say?

Several epidemiology studies suggest that host genetic factors play important roles in susceptibility, protection and progression of tuberculosis infection. Here we have reviewed the implications of some genetic polymorphisms in pathways related to tuberculosis susceptibility, severity and development. Large case-control studies examining single-nucleotide polymorphisms (SNPs) in genes have been performed in tuberculosis patients in some countries. Polymorphisms in natural resistance-associated macrophage protein 1 (NRAMP1), toll-like receptor 2 (TLR2), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), interleukin-1 receptor antagonist (IL-1RA), IL-10, vitamin D receptor (VDR), dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN), monocyte chemoattractant protein-1 (MCP-1), nucleotide oligomerization binding domain 2 (NOD2), interferon-gamma (IFN-γ), inducible nitric oxide synthase (iNOS), mannose-binding lectin (MBL) and surfactant proteins A (SP-A) have been reviewed. These genes have been variably associated with tuberculosis infection and there is strong evidence indicating that host genetic factors play critical roles in tuberculosis susceptibility, severity and development.