Structure-Guided Discovery of PD-1/PD-L1 Interaction Inhibitors: Peptide Design, Screening, and Optimization via Computation-Aided Phage Display Engineering.

Cancer immunotherapy harnesses the immune system to combat tumors and has emerged as a major cancer treatment modality. The PD-1/PD-L1 immune checkpoint modulates interactions between tumor cells and T cells and has been extensively targeted in cancer immunotherapy. However, the monoclonal antibodies known to target this immune checkpoint have considerable side effects, and novel PD-1/PD-L1 inhibitors are therefore required. Herein, a peptide inhibitor to disrupt PD-1/PD-L1 interactions was designed through structure-driven phage display engineering coupled to computational modification and optimization. BetaPb, a novel peptide library constructed by using the known structure of PD-1/PD-L, was used to develop inhibitors against the immune checkpoint, and specific peptides with high affinity toward PD-1 were screened through enzyme-linked immunosorbent assays, homogeneous time-resolved fluorescence, and biolayer interferometry. A potential inhibitor, B8, was preliminarily screened through biopanning. The binding affinity of B8 toward PD-1 was confirmed through computation-aided optimization. Assessment of B8 variants (B8.1, B8.2, B8.3, B8.4, and B8.5) demonstrated their attenuation of PD-1/PD-L1 interactions. B8.4 exhibited the strongest attenuation efficiency at a half-maximal effective concentration of 0.1 µM and the strongest binding affinity to PD-1 (equilibrium dissociation constant = 0.1 µM). B8.4 outperformed the known PD-1/PD-L1 interaction inhibitor PL120131 in disrupting PD-1/PD-L1 interactions, revealing that B8.4 has remarkable potential for modification to yield an antitumor agent. This study provides valuable information for the future development of peptide-based drugs, therapeutics, and immunotherapies for cancer.

Upregulation of ACE2 and TMPRSS2 by particulate matter and idiopathic pulmonary fibrosis: a potential role in severe COVID-19.

BACKGROUND: Air pollution exposure and idiopathic pulmonary fibrosis (IPF) cause a poor prognosis after SARS-CoV-2 infection, but the underlying mechanisms are not well explored. Angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) are the keys to the entry of SARS-CoV-2. We therefore hypothesized that air pollution exposure and IPF may increase the expression of ACE2 and TMPRSS2 in the lung alveolar region. We measured their expression levels in lung tissues of control non-IPF and IPF patients, and used murine animal models to study the deterioration of IPF caused by particulate matter (PM) and the molecular pathways involved in the expression of ACE2 and TMPRSS2. RESULTS: In non-IPF patients, cells expressing ACE2 and TMPRSS2 were limited to human alveolar cells. ACE2 and TMPRSS2 were largely upregulated in IPF patients, and were co-expressed by fibroblast specific protein 1 (FSP-1) + lung fibroblasts in human pulmonary fibrotic tissue. In animal models, PM exposure increased the severity of bleomycin-induced pulmonary fibrosis. ACE2 and TMPRSS2 were also expressed in FSP-1+ lung fibroblasts in bleomycin-induced pulmonary fibrosis, and when combined with PM exposure, they were further upregulated. The severity of pulmonary fibrosis and the expression of ACE2 and TMPRSS2 caused by PM exposure were blocked by deletion of KC, a murine homologue of IL-8, or treatment with reparixin, an inhibitor of IL-8 receptors CXCR1/2. CONCLUSIONS: These data suggested that risk of SARS-CoV-2 infection and COVID-19 disease severity increased by air pollution exposure and underlying IPF. It can be mediated through upregulating ACE2 and TMPRSS2 in pulmonary fibroblasts, and prevented by blocking the IL-8/CXCR1/2 pathway.

Involvement of collagen XVII in pluripotency gene expression and metabolic reprogramming of lung cancer stem cells.

BACKGROUND: Recent advancements in cancer biology field suggest that glucose metabolism is a potential target for cancer treatment. However, little if anything is known about the metabolic profile of cancer stem cells (CSCs) and the related underlying mechanisms. METHODS: The metabolic phenotype in lung CSC was first investigated. The role of collagen XVII, a putative stem cell or CSC candidate marker, in regulating metabolic reprogramming in lung CSC was subsequently studied. Through screening the genes involved in glycolysis, we identified the downstream targets of collagen XVII that were involved in metabolic reprogramming of lung CSCs. Collagen XVII and its downstream targets were then used to predict the prognosis of lung cancer patients. RESULTS: We showed that an aberrant upregulation of glycolysis and oxidative phosphorylation in lung CSCs is associated with the maintenance of CSC-like features, since blocking glycolysis and oxidative phosphorylation reduces sphere formation, chemoresistance, and tumorigenicity. We also showed that the Oct4-hexokinase 2 (HK2) pathway activated by collagen XVII-laminin-332 through FAK-PI3K/AKT-GSB3ß/ß-catenin activation induced the upregulation of glycolysis and maintenance of CSC-like features. Finally, we showed that collagen XVII, Oct4, and HK2 could be valuable markers to predict the prognosis of lung cancer patients. CONCULSIONS: These data suggest the Oct4-HK2 pathway regulated by collagen XVII plays an important role in metabolic reprogramming and maintenance of CSC-like features in lung CSCs, which may aid in the development of new strategies in cancer treatment.

High metabolic rate and stem cell characteristics of esophageal cancer stem-like cells depend on the Hsp27-AKT-HK2 pathway.

Tumor progression with chemoresistance and local recurrence is commonly happened during treatment of esophageal squamous cell carcinoma (ESCC). Cancer stem cells (CSC) may respond for tumor progression. However, there are few reports regarding metabolism of esophageal CSCs with clinical correlation. In this work, we demonstrated that ESCC cell lines in spheroid culture display CSC phenotypes, including increased ALDH activity, chemoresistance and tumor initiation, which are dependent on Hsp27 activation. Esophageal CSCs also exhibit reprogrammed metabolic features particularly higher glycolysis and oxidative phosphorylation, which are regulated via the Hsp27-AKT-HK2 pathway. Moreover, HK2 is required for maintenance of CSC phenotypes. Inhibition of CSC metabolism reduces cell growth and tumor formation. Clinically, patients who underwent surgical resection for esophageal cancer, and displayed overexpression of both Hsp27 and HK2, had the worst prognosis of all expression types. In conclusion, stem cells features and aberrant metabolic reprogramming of esophageal CSCs depend on the Hsp27-AKT-HK2 pathway. Targeting Hsp27 and HK2 could be novel therapeutic strategy for treating esophageal cancer and warrants further investigation.

Particulate Matter Increases the Severity of Bleomycin-Induced Pulmonary Fibrosis through KC-Mediated Neutrophil Chemotaxis.

BACKGROUND: Although particular matter (PM) increases incidence and severity of idiopathic pulmonary fibrosis, the underlying mechanism remains elusive. METHODS: The effects of PM were evaluated in a murine model of bleomycin-induced pulmonary fibrosis. Mice were divided into four groups, receiving: (1) Saline (control), (2) bleomycin, (3) PM, or (4) bleomycin plus PM (Bleo+PM). Additional groups of Bleo+PM mice were treated with sivelestat (an inhibitor of neutrophil elastase) or reparixin (a C-X-C motif chemokine receptor 2 antagonist), or were genetically modified with keratinocyte chemoattractant (KC) deletion. RESULTS: Pulmonary fibrosis was not observed in the control or PM groups. Bleomycin induced pulmonary fibrosis within 14 days. The Bleo+PM group showed worse pulmonary fibrosis when compared to the bleomycin group. Analyses of immune cell profile and chemokine/cytokine concentrations at day 2-bronchoalveolar lavage fluid (BALF) revealed that the Bleo+PM group had increased neutrophil number and elastase level and KC concentration compared to the bleomycin group. Neutrophil elastase activated the Smad2/Smad3/α-SMA pathway to induce collagen deposition, while sivelestat abrogated the increased severity of pulmonary fibrosis caused by PM. Chemotaxis assay revealed that BALF of the Bleo+PM group recruited neutrophil, which was dependent on KC. Further, genetic KC deletion or pharmaceutical inhibition of KC binding to CXCR2 with reparixin ameliorated the PM-induced increased severity of pulmonary fibrosis. CONCLUSIONS: These data provide evidence that the PM-induced increased severity of pulmonary fibrosis depends on KC-mediated neutrophil chemotaxis and give additional mechanic insight that will aid in the development of therapeutic strategies.

Epstein-Barr virus LMP2A suppresses MHC class II expression by regulating the B-cell transcription factors E47 and PU.1.

Oncogenic Epstein-Barr virus (EBV) uses various approaches to escape host immune responses and persist in B cells. Such persistent infections may provide the opportunity for this virus to initiate tumor formation. Using EBV-immortalized lymphoblastoid cell lines (LCLs) as a model, we found that the expression of major histocompatibility complex (MHC) class II and CD74 in B cells is repressed after EBV infection. Class II transactivator (CIITA) is the master regulator of MHC class II-related genes. As expected, CIITA was downregulated in LCLs. We showed that downregulation of CIITA is caused by EBV latent membrane protein 2A (LMP2A) and driven by the CIITA-PIII promoter. Furthermore, we demonstrated that LMP2A-mediated E47 and PU.1 reduction resulted in CIITA suppression. Mechanistically, the LMP2A immunoreceptor tyrosine-based activation motif was critical for the repression of E47 and PU.1 promoter activity via Syk, Src, and the phosphatidylinositol 3-kinase/Akt pathway. Elimination of LMP2A in LCLs using a shLMP2A approach showed that the expression levels of E47, PU.1, CIITA, MHC class II, and CD74 are reversed. These data indicated that the LMP2A may reduce MHC class II expression through interference with the E47/PU.1-CIITA pathway. Finally, we demonstrated that MHC class II may be detected in tonsils and EBV-negative Hodgkin disease but not in EBV-associated posttransplant lymphoproliferative disease and Hodgkin disease.

Maintenance of Epstein-Barr Virus Latent Status by a Novel Mechanism, Latent Membrane Protein 1-Induced Interleukin-32, via the Protein Kinase Cδ Pathway.

UNLABELLED: Epstein-Barr virus (EBV), an oncogenic herpesvirus, has the potential to immortalize primary B cells into lymphoblastoid cell lines (LCLs) in vitro. During immortalization, several EBV products induce cytokines or chemokines, and most of these are required for the proliferation of LCLs. Interleukin-32 (IL-32), a recently discovered proinflammatory cytokine, is upregulated after EBV infection, and this upregulation is detectable in all LCLs tested. EBV latent membrane protein 1 (LMP1) is responsible for inducing IL-32 expression at the mRNA and protein levels. Mechanistically, we showed that this LMP1 induction is provided by the p65 subunit of NF-κB, which binds to and activates the IL-32 promoter. Furthermore, the short hairpin RNA (shRNA)-mediated depletion of endogenous LMP1 and p65 in LCLs suppressed IL-32 expression, further suggesting that LMP1 is the key factor that stimulates IL-32 in LCLs via the NF-κB p65 pathway. Functionally, knockdown of IL-32 in LCLs elicits viral reactivation and affects cytokine expression, but it has no impact on cell proliferation and apoptosis. Of note, we reveal the mechanism whereby IL-32 is involved in the maintenance of EBV viral latency by inactivation of Zta promoter activity. This atypical cytoplasmic IL-32 hijacks the Zta activator protein kinase Cδ (PKCδ) and inhibits its translocation from the cytoplasm to the nucleus, where PKCδ binds to the Zta promoter and activates lytic cycle progression. These novel findings reveal that IL-32 is involved in the maintenance of EBV latency in LCLs. This finding may provide new information to explain how EBV maintains latency, in addition to viral chromatin structure and epigenetic modification. IMPORTANCE: EBV persists in two states, latency and lytic replication, which is a unique characteristic of human infections. So far, little is known about how herpesviruses maintain latency in particular tissues or cell types. EBV is an excellent model to study this question because more than 90% of people are latently infected. EBV can immortalize primary B cells into lymphoblastoid cell lines in vitro. Expression of IL-32, a novel atypical cytoplasmic proinflammatory cytokine, increased after infection. The expression of IL-32 was controlled by LMP1. In investigating the regulatory mechanism, we demonstrated that the p65 subunit of NF-κB is required for this upregulation. Of note, the important biological activity of IL-32 was to trap protein kinase Cδ in the cytoplasm and prevent it from binding to the Zta promoter, which is the key event for EBV reaction. So, the expression of LMP1-induced IL-32 plays a role in the maintenance of EBV latency.

IL-6 enriched lung cancer stem-like cell population by inhibition of cell cycle regulators via DNMT1 upregulation.

Tumors are influenced by a microenvironment rich in inflammatory cytokines, growth factors and chemokines, which may promote tumor growth. Interleukin-6 (IL-6) is a multifunctional cytokine and known as a regulator of immune and inflammation responses. IL-6 has also been reported to be associated with tumor progression and chemoresistance in different types of cancers. In our study, we demonstrated that IL-6 enriches the properties of lung cancer stem-like cells in A549 lung cancer cells cultured in spheroid medium. IL-6 also promotes sphere formation and stem-like properties of A549 cells by enhancing cell proliferation. Methylation-specific polymerase chain reaction (PCR) was performed and revealed that IL-6 increased methylation of p53 and p21 in A549 cancer cells. Western blot analysis and quantitative real-time PCR demonstrated that IL-6 increased the expression of DNA methyltransferase 1 (DNMT1) in A549 cells cultured in spheroid medium, but not the expression of DNMT3a or DNMT3b. Knockdown of DNMT1 eliminated IL-6-mediated hypermethylation of cell cycle regulators and enrichment of lung cancer stem-like properties. In conclusion, our study, for the first time, shows that the IL-6/JAK2/STAT3 pathway upregulates DNMT1 and enhances cancer initiation and lung cancer stem cell (CSC) proliferation by downregulation of p53 and p21 resulting from DNA hypermethylation. Upon blockage of the IL-6/JAK2/STAT3 pathway and inhibition of DNMT1, the proliferation of lung CSCs was reduced and their formation of spheres and ability to initiate tumor growth were decreased. These data suggest that targeting of the IL-6/JAK2/STAT3 signaling pathway and DNMT1 may become important strategies for treating lung cancer.

Autocrine CCL3 and CCL4 induced by the oncoprotein LMP1 promote Epstein-Barr virus-triggered B cell proliferation.

Epstein-Barr virus (EBV) alters the regulation and expression of a variety of cytokines in its host cells to modulate host immune surveillance and facilitate viral persistence. Using cytokine antibody arrays, we found that, in addition to the cytokines reported previously, two chemotactic cytokines, CCL3 and CCL4, were induced in EBV-infected B cells and were expressed at high levels in all EBV-immortalized lymphoblastoid cell lines (LCLs). Furthermore, EBV latent membrane protein 1 (LMP1)-mediated Jun N-terminal protein kinase activation was responsible for upregulation of CCL3 and CCL4. Inhibition of CCL3 and CCL4 in LCLs using a short hairpin RNA approach or by neutralizing antibodies suppressed cell proliferation and caused apoptosis, indicating that autocrine CCL3 and CCL4 are required for LCL survival and growth. Importantly, significant amounts of CCL3 were detected in EBV-positive plasma from immunocompromised patients, suggesting that EBV modulates this chemokine in vivo. This study reveals the regulatory mechanism and a novel function of CCL3 and CCL4 in EBV-infected B cells. CCL3 might be useful as a therapeutic target in EBV-associated lymphoproliferative diseases and malignancies.

Selenite selectively kills lung fibroblasts to treat bleomycin-induced pulmonary fibrosis.

BACKGROUND: Interstitial lung disease (ILD) treatment is a critical unmet need. Selenium is an essential trace element for human life and an antioxidant that activates glutathione, but the gap between its necessity and its toxicity is small and requires special attention. Whether selenium can be used in the treatment of ILD remains unclear. METHODS: We investigated the prophylactic and therapeutic effects of selenite, a selenium derivative, in ILD using a murine model of bleomycin-induced idiopathic pulmonary fibrosis (IPF). We further elucidated the underlying mechanism using in vitro cell models and examined their relevance in human tissue specimens. The therapeutic effect of selenite in bleomycin-administered mice was assessed by respiratory function and histochemical changes. Selenite-induced apoptosis and reactive oxygen species (ROS) production in murine lung fibroblasts were measured. RESULTS: Selenite, administered 1 day (inflammation phase) or 8 days (fibrotic phase) after bleomycin, prevented and treated deterioration of lung function and pulmonary fibrosis in mice. Mechanistically, selenite inhibited the proliferation and induced apoptosis of murine lung fibroblasts after bleomycin treatment both in vitro and in vivo. In addition, selenite upregulated glutathione reductase (GR) and thioredoxin reductase (TrxR) in murine lung fibroblasts, but not in lung epithelial cells, upon bleomycin treatment. GR and TrxR inhibition eliminates the therapeutic effects of selenite. Furthermore, we found that GR and TrxR were upregulated in the human lung fibroblasts of IPF patient samples. CONCLUSIONS: Selenite induces ROS production and apoptosis in murine lung fibroblasts through GR and TrxR upregulation, thereby providing a therapeutic effect in bleomycin-induced IPF.

Stable laminar shear stress induces G1 cell cycle arrest and autophagy in urothelial carcinoma by a torque sensor-coupled cone-and-plate device.

The microenvironments of urinary systems play crucial roles in the development and metastasis of cancers due to their generation of complex temporal and spatial fluidic profiles. Because of their versatility in creating desired biomimetic flow, cone-and-plate bioreactors offer great potential for bladder cancer research. In this study, we construct a biocompatible cone-and-plate device coupled with a torque sensor, enabling the application and real-time monitoring of stable shear stress up to 50 dyne/cm². Under a stable shear stress stimulation at 12 dyne/cm2, bladder cancer cell BFTC-905 is arrested at the G1 phase with decreased cell proliferation after 24-hour treatment. This effect is associated with increased cyclin-dependent kinase inhibitors p21 and p27, inhibiting cyclin D1/CDK4 complex with dephosphorylation of serine 608 on the retinoblastoma protein. Consequently, an increase in cyclin D3 and decreases in cyclin A2 and cyclin E2 are observed. Moreover, we demonstrate that the shear stress stimulation upregulates the expression of autophagy-related proteins Beclin-1, LC3B-I and LC3B-II, while caspase cleavages are not activated under the same condition. The design of this system and its application shed new light on flow-induced phenomena in the study of urothelial carcinomas.

Structure-guided identification and characterization of potent inhibitors targeting PhoP and MtrA to combat mycobacteria.

Mycobacteria are causative agents of tuberculosis (TB), which is a global health concern. Drug-resistant TB strains are rapidly emerging, thereby necessitating the urgent development of new drugs. Two-component signal transduction systems (TCSs) are signaling pathways involved in the regulation of various bacterial behaviors and responses to environmental stimuli. Applying specific inhibitors of TCSs can disrupt bacterial signaling, growth, and virulence, and can help combat drug-resistant TB. We conducted a comprehensive pharmacophore-based inhibitor screening and biochemical and biophysical examinations to identify, characterize, and validate potential inhibitors targeting the response regulators PhoP and MtrA of mycobacteria. The constructed pharmacophore model Phar-PR-n4 identified effective inhibitors of formation of the PhoP-DNA complex: ST132 (IC50 = 29 ± 1.6 µM) and ST166 (IC50 = 18 ± 1.3 µM). ST166 (KD = 18.4 ± 4.3 µM) and ST132 (KD = 14.5 ± 0.1 µM) strongly targeted PhoP in a slow-on, slow-off manner. The inhibitory potency and binding affinity of ST166 and ST132 for MtrAC were comparable to those of PhoP. Structural analyses and molecular dynamics simulations revealed that ST166 and ST132 mainly interact with the α8-helix and C-terminal ß-hairpin of PhoP, with functionally essential residue hotspots for structure-based inhibitor optimization. Moreover, ST166 has in vitro antibacterial activity against Macrobacterium marinum. Thus, ST166, with its characteristic 1,2,5,6-tetrathiocane and terminal sulphonic groups, has excellent potential as a candidate for the development of novel antimicrobial agents to combat pathogenic mycobacteria.

Requirement for LMP1-induced RON receptor tyrosine kinase in Epstein-Barr virus-mediated B-cell proliferation.

EBV, an oncogenic human herpesvirus, can transform primary B lymphocytes into immortalized lymphoblastoid cell lines (LCLs) through multiple regulatory mechanisms. However, the involvement of protein tyrosine kinases in the infinite proliferation of B cells is not clear. In this study, we performed kinase display assays to investigate this subject and identified a specific cellular target, Recepteur d'Origine Nantais (RON) tyrosine kinase, expressed in LCLs but not in primary B cells. Furthermore, we found that latent membrane protein 1 (LMP1), an important EBV oncogenic protein, enhanced RON expression through its C-terminal activation region-1 (CTAR1) by promoting NF-κB binding to the RON promoter. RON knockdown decreased the proliferation of LCLs, and transfection with RON compensated for the growth inhibition caused by knockdown of LMP1. Immunohistochemical analysis revealed a correlation between LMP1 and RON expression in biopsies from posttransplantation lymphoproliferative disorder (PTLD), suggesting that LMP1-induced RON expression not only is essential for the growth of LCLs but also may contribute to the pathogenesis of EBV-associated PTLD. Our study is the first to reveal the impact of RON on the proliferation of transformed B cells and to suggest that RON may be a novel therapeutic target for EBV-associated lymphoproliferative diseases.

Interplay between PKCδ and Sp1 on histone deacetylase inhibitor-mediated Epstein-Barr virus reactivation.

Epstein-Barr virus (EBV) undergoes latent and lytic replication cycles, and its reactivation from latency to lytic replication is initiated by expression of the two viral immediate-early transactivators, Zta and Rta. In vitro, reactivation of EBV can be induced by anti-immunoglobulin, tetradecanoyl phorbol acetate, and histone deacetylase inhibitor (HDACi). We have discovered that protein kinase C delta (PKCδ) is required specifically for EBV reactivation by HDACi. Overexpression of PKCδ is sufficient to induce the activity of the Zta promoter (Zp) but not of the Rta promoter (Rp). Deletion analysis revealed that the ZID element of Zp is important for PKCδ activation. Moreover, the Sp1 putative sequence on ZID is essential for PKCδ-induced Zp activity, and the physiological binding of Sp1 on ZID has been confirmed. After HDACi treatment, activated PKCδ can phosphorylate Sp1 at serine residues and might result in dissociation of the HDAC2 repressor from ZID. HDACi-mediated HDAC2-Sp1 dissociation can be inhibited by the PKCδ inhibitor, Rotterlin. Furthermore, overexpression of HDAC2 can suppress the HDACi-induced Zp activity. Consequently, we hypothesize that HDACi induces PKCδ activation, causing phosphorylation of Sp1, and that the interplay between PKCδ and Sp1 results in the release of HDAC2 repressor from Zp and initiation of Zta expression.

p53 and Sp1 cooperate to regulate the expression of Epstein-Barr viral Zta protein.

Epstein-Barr virus (EBV) belongs to the gammaherpesvirus family. To produce infectious progeny, EBV reactivates from latency into the lytic cycle by expressing the determinative lytic transactivator, Zta. In the presence of histone deacetylase inhibitor (HDACi), p53 is a prerequisite for the initiation of the EBV lytic cycle by facilitating the expression of Zta. In this study, a serial mutational analysis of Zta promoter (Zp) indicated an important role for the ZID element in responding to HDACi induction and p53 binds to this ZID element together with Sp1, a universal transcription factor. Abolition of the DNA-binding ability of Sp1 reduces the inducibility of ZID by HDACi and also reduces the amount of p53 binding to ZID. Finally, it was shown that EBV in p53-positive-lymphoblastoid cell lines (LCLs) can enter into the lytic cycle spontaneously; however, knockdown of p53 in LCLs leads to retardation of EBV reactivation.

Glutathione peroxidase 4 expression predicts poor overall survival in patients with resected lung adenocarcinoma.

This study aimed to evaluate the protein expression of glutathione peroxidase 4 (GPX4) in resected non-small cell lung cancer (NSCLC). The clinical relevance and prognostic significance of GPX4 expression were analyzed. We reviewed patients with resected NSCLCs at Taipei Veterans General Hospital between September 2002 and January 2018. Available paraffin-embedded specimens were retrieved for immunohistochemistry (IHC) staining to detect GPX4 expression. The cutoff value for defining GPX4 positivity was determined according to the percentage of tumor stained in the microscopic field. The correlation between immune expression, clinicopathologic data, overall survival (OS), and disease-free survival (DFS) were analyzed. A total of 265 NSCLC specimens were retrieved for IHC staining. GPX4 expression positive was in 192 (72.5%) according to a cutoff value of 5%. GPX4 was a significant prognostic factor for OS and DFS on multivariate analysis at both 5% and 25% cutoff values. GPX4 expression was associated with poor OS and DFS, especially in lung adenocarcinoma (p = 0.008, and 0.027, respectively). In conclusions, IHC analysis revealed that GPX4 expression was associated with poor survival outcomes in patients with resected lung adenocarcinoma. Further research is needed to understand the role of GPX4 in tumorigenesis and the underlying mechanism responsible for survival outcomes in patients with resected lung adenocarcinoma.

Regulation of microtubule dynamics through phosphorylation on stathmin by Epstein-Barr virus kinase BGLF4.

Stathmin is an important microtubule (MT)-destabilizing protein, and its activity is differently attenuated by phosphorylation at one or more of its four phosphorylatable serine residues (Ser-16, Ser-25, Ser-38, and Ser-63). This phosphorylation of stathmin plays important roles in mitotic spindle formation. We observed increasing levels of phosphorylated stathmin in Epstein-Barr virus (EBV)-harboring lymphoblastoid cell lines (LCLs) and nasopharyngeal carcinoma (NPC) cell lines during the EBV lytic cycle. These suggest that EBV lytic products may be involved in the regulation of stathmin phosphorylation. BGLF4 is an EBV-encoded kinase and has similar kinase activity to cdc2, an important kinase that phosphorylates serine residues 25 and 38 of stathmin during mitosis. Using an siRNA approach, we demonstrated that BGLF4 contributes to the phosphorylation of stathmin in EBV-harboring NPC. Moreover, we confirmed that BGLF4 interacts with and phosphorylates stathmin using an in vitro kinase assay and an in vivo two-dimensional electrophoresis assay. Interestingly, unlike cdc2, BGLF4 was shown to phosphorylate non-proline directed serine residues of stathmin (Ser-16) and it mediated phosphorylation of stathmin predominantly at serines 16, 25, and 38, indicating that BGLF4 can down-regulate the activity of stathmin. Finally, we demonstrated that the pattern of MT organization was changed in BGLF4-expressing cells, possibly through phosphorylation of stathmin. In conclusion, we have shown that a viral Ser/Thr kinase can directly modulate the activity of stathmin and this contributes to alteration of cellular MT dynamics and then may modulate the associated cellular processes.

Esophageal Cancer Stem-like Cells Resist Ferroptosis-Induced Cell Death by Active Hsp27-GPX4 Pathway.

Cancer stem cells (CSCs), a subpopulation of cancer cells responsible for tumor initiation and treatment failure, are more susceptible to ferroptosis-inducing agents than bulk cancer cells. However, regulatory pathways controlling ferroptosis, which can selectively induce CSC death, are not fully understood. Here, we demonstrate that the CSCs of esophageal squamous carcinoma cells enriched by spheroid culture have increased intracellular iron levels and lipid peroxidation, thereby increasing exposure to several products of lipid peroxidation, such as MDA and 4-HNE. However, CSCs do not reduce cell viability until glutathione is depleted by erastin treatment. Mechanistic studies revealed that damage from elevated lipid peroxidation is avoided through the activation of Hsp27, which upregulates GPX4 and thereby rescues CSCs from ferroptosis-induced cell death. Our results also revealed a correlation between phospho-Hsp27 and GPX4 expression levels and poor prognosis in patients with esophageal cancer. Together, these data indicate that targeting Hsp27 or GPX4 to block this intrinsic protective mechanism against ferroptosis is a potential treatment strategy for eradicating CSC in esophageal squamous cell carcinoma.

Collagen XVII/laminin-5 activates epithelial-to-mesenchymal transition and is associated with poor prognosis in lung cancer.

Epithelial-to-mesenchymal transition (EMT) is associated with tumor metastasis and tumorigenesis in lung cancer stem-like cells (CSCs). However, the exact mechanism underlying this is not clear. We used microarray analysis to identify candidate genes responsible for EMT in spheroid and monolayer cultures of lung cancer cells. We found increased expression of a variety of adhesion molecules in CSCs. One of these molecules, Collagen XVII (Col XVII), was demonstrated to be required for maintenance of EMT phenotypes and metastasis ability in lung CSCs. We showed that Col XVII stabilized laminin-5 to activate the FAK/AKT/GSK3ß pathway, thereby suppressing Snail ubiquitination-degradation. The function of Col XVII was mainly dependent on shedding by ADAM9 and ADAM10. Patients who underwent surgical resection for lung cancer, and displayed overexpression of both Col XVII and laminin-5, had the worst prognosis of all expression types. Moreover, blockage of the Col XVII/laminin-5 pathway reduced the EMT phenotypes of lung CSCs in vitro and decreased the potential of lung metastasis in vivo. Our findings suggested that targeting Col XVII and laminin-5 could be novel therapeutic strategies for treating lung cancer patients, and warrant further investigation.

Involvement of ER stress, PI3K/AKT activation, and lung fibroblast proliferation in bleomycin-induced pulmonary fibrosis.

Pulmonary fibrosis is characterized by fibroblast proliferation and extracellular matrix remodelling, leading to respiratory insufficiency. The mechanisms underlying this progressive and devastating disease remain unclear. Conditions that can impair the function of the endoplasmic reticulum (ER) cause accumulation of unfolded or misfolded proteins, resulting in ER stress and activation of the unfolded protein response (UPR). ER stress has been implicated in many conditions including cancer, diabetes, obesity, and inflammation. It is also involved in lung fibrosis, through myofibroblastic differentiation of fibroblasts; however, the precise role of ER stress in lung fibrosis is unknown. The current study aimed to investigate the underlying mechanisms of ER stress inhibitors in the treatment of bleomycin-induced lung fibrosis. We demonstrated that bleomycin can activate ER stress associated proteins, including GRP78, CHOP, and ATF-4, both in vitro and in vivo. PI3K/AKT acts upstream of ER stress to affect lung fibroblast proliferation, resulting in bleomycin-induced pulmonary fibrosis. Treatment with ER stress inhibitors or a PI3K inhibitor caused a reduction in fibroblast proliferation and improved pulmonary function. The relationship between PI3K/AKT/mTOR and ER stress in pulmonary fibrosis, and the application of PI3K inhibitors and ER stress inhibitors in the treatment of pulmonary fibrosis require further investigation.