Upregulation of HLA-II related to LAG-3+CD4+ T cell infiltration is associated with patient outcome in human glioblastoma.

Glioblastoma (GBM) is the most common malignant diffuse glioma of the brain. Although immunotherapy with immune checkpoint inhibitors (ICIs), such as programmed cell death protein (PD)-1/PD ligand-1 inhibitors, has revolutionized the treatment of several cancers, the clinical benefit in GBM patients has been limited. Lymphocyte-activation gene 3 (LAG-3) binding to human leukocyte antigen-II (HLA-II) plays an essential role in triggering CD4+ T cell exhaustion and could interfere with the efficiency of anti-PD-1 treatment; however, the value of LAG-3-HLA-II interactions in ICI immunotherapy for GBM patients has not yet been analyzed. Therefore, we aimed to investigate the expression and regulation of HLA-II in human GBM samples and the correlation with LAG-3+CD4+ T cell infiltration. Human leukocyte antigen-II was highly expressed in GBM and correlated with increased LAG-3+CD4+ T cell infiltration in the stroma. Additionally, HLA-IIHighLAG-3High was associated with worse patient survival. Increased interleukin-10 (IL-10) expression was observed in GBM, which was correlated with high levels of HLA-II and LAG-3+ T cell infiltration in stroma. HLA-IIHighIL-10High GBM associated with LAG-3+ T cells infiltration synergistically showed shorter overall survival in patients. Combined anti-LAG-3 and anti-IL-10 treatment inhibited tumor growth in a mouse brain GL261 tumor model. In vitro, CD68+ macrophages upregulated HLA-II expression in GBM cells through tumor necrosis factor-α (TNF-α). Blocking TNF-α-dependent inflammation inhibited tumor growth in a mouse GBM model. In summary, T cell-tumor cell interactions, such as LAG-3-HLA-II, could confer an immunosuppressive environment in human GBM, leading to poor prognosis in patients. Therefore, targeting the LAG-3-HLA-II interaction could be beneficial in ICI immunotherapy to improve the clinical outcome of GBM patients.

The underlying molecular mechanism of ciliated epithelium dysfunction and TGF-ß signaling in children with congenital pulmonary airway malformations.

The aim of this study was to investigate the variation in gene expression in the complete transcripts of Congenitalpulmonary airwaymalformation (CPAM) of the lung using Next Generation Sequencing (NGS) technology. There were 20 cases involving children with CPAM were used for selection of study sample. NGS was used to establish RNA-Seq libraries for the two groups of samples separately, and both groups were conducted to differential expression analysis and Gene Ontology (GO) functional enrichment analysis. The pathways of the differential genes were analyzed to find the enriched target pathways. A total of 592 genes were expressed with significant differences (CPAM vs. normal tissue, P < 0.05). GO functional analysis of DEGs indicated that abnormal ciliary function played a role in the development of CPAM. Subsequently, analysis of these genes pathways showed the TGF-ß signaling pathway was significantly enriched. Finally, the results of immunohistochemical analysis of some DEGs showed that a significant reduction in the expression of SMAD6, a gene related to the TGF-ß signaling pathway, led to abnormal activation of the pathway. TGF-ß signaling pathway involved in the evolution of the disease obtained by DEGs enrichment pathway analysis. SMAD6, a gene involved in this pathway, might be a potential biomarker for the diagnosis and treatment of CPAM.

Inhibition of N-myristoyltransferase activity promotes androgen receptor degradation in prostate cancer.

BACKGROUND: Even though prostate cancer (PCa) patients initially respond to androgen deprivation therapy, some will eventually develop castration resistant prostate cancer (CRPC). Androgen receptor (AR) mediated cell signaling is a major driver in the progression of CRPC while only a fraction of PCa becomes AR negative. This study aimed to understand the regulation of AR levels by N-myristoyltransferase in PCa cells. METHODS: Two enantiomers, (1S,2S)- d-NMAPPD and (1R,2R)- d-NMAPPD (LCL4), were characterized by various methods (1 H and 13 C NMR, UHPLC, high-resolution mass spectra, circular dichroism) and evaluated for the ability to bind to N-myristoyltransferase 1 (NMT1) using computational docking analysis. structure-activity relationship analysis of these compounds led to the synthesis of (1R,2R)-LCL204 and evaluation as a potential NMT1 inhibitor utilizing the purified full length NMT1 enzyme. The NMT inhibitory activity wase determined by Click chemistry and immunoblotting. Regulation of NMT1 on tumor growth was evaluated in a xenograft tumor model. RESULTS: (1R,2R)- d-NMAPPD, but not its enantiomer (1S,2S)- d-NMAPPD, inhibited NMT1 activity and reduced AR protein levels. (1R,2R)-LCL204, a derivative of (1R,2R)- d-NMAPPD, inhibited global protein myristoylation. It also suppressed protein levels, nuclear translocation, and transcriptional activity of AR full-length or variants in PCa cells. This was due to enhanced ubiquitin and proteasome-mediated degradation of AR. Knockdown of NMT1 levels inhibited tumor growth and proliferation of cancer cells. CONCLUSION: Inhibitory efficacy on N-myristoyltransferase activity by d-NMAPPD is stereospecific. (1R,2R)-LCL204 reduced global N-myristoylation and androgen receptor protein levels at low micromolar concentrations in prostate cancer cells. pharmacological inhibition of NMT1 enhances ubiquitin-mediated proteasome degradation of AR. This study illustrates a novel function of N-myristoyltransferase and provides a potential strategy for treatment of CRPC.

Ochratoxin A-enhanced glycolysis induces inflammatory responses in human gastric epithelium cells through mTOR/HIF-1α signaling pathway.

Ochratoxin A (OTA) is a mycotoxin commonly found in several food commodities worldwide with potential nephrotoxic, hepatotoxic and carcinogenic effects. We previously showed for the first time that OTA treatment enhanced glycolysis in human gastric epithelium (GES-1) cells in vitro. Here, we found that OTA exposure activated inflammatory responses, evidenced by increasing of NF-κB signaling pathway-related protein (p-p65 and p-IκBα) expressions and elevating of inflammatory cytokine (IL-1ß and IL-6) mRNA expressions in GES-1 cells. To elucidate the role of glycolysis in inflammatory effects triggered by OTA, we pretreated GES-1 cells with glycolysis inhibitor (2-deoxy-D-glucose, 2-DG) before OTA exposure. The result showed that 2-DG reduced the protein expressions of p-p65 and p-IκBα and alleviated the mRNA expressions of inflammatory cytokines in OTA-treated GES-1 cells. Furthermore, OTA activated the mTOR/HIF-1α pathway by increasing the protein expressions of p-mTOR, p-eIF4E and HIF-1α, and inhibition of mTOR with rapamycin or silencing HIF-1α with siRNA significantly attenuated OTA-enhanced glycolysis by reducing glycolysis related genes and thereby decreasing inflammatory effects of GES-1 cells. These results demonstrate that OTA activates inflammatory responses in GES-1 cells and this is controlled by mTOR/HIF-1α pathway-mediated glycolysis enhancement. Our findings provide a novel mechanistic view into OTA-induced gastric cytotoxicity.

Expression, clinicopathological significance, and prognostic potential of AMPK, p-AMPK, PGC-1α, and TFAM in astrocytomas.

AMP-activated protein kinase (AMPK) is a sensor of energy status that maintains cellular energy homeostasis. Activation of AMPK enhances the expression of proliferator-activated receptor γ coactivator 1α (PGC1-α) and subsequently activates mitochondrial transcription factor A (TFAM) to regulate mitochondrial oxidative respiratory function. The possible functions of AMPK, p-AMPK, PGC-1α, and TFAM and their interactions in astrocytomas are not known. Here, the levels, clinicopathological characteristics, and prognostic potential of AMPK, p-AMPK, PGC-1α, and TFAM expression levels in astrocytomas were evaluated. The results showed that levels of AMPK, p-AMPK, PGC-1α, and TFAM expression was increased in astrocytomas. Strong correlations were observed between AMPK, p-AMPK, PGC-1α, and TFAM expression in patients with astrocytomas. The analysis indicated that the levels of AMPK, p-AMPK, PGC-1α, and TFAM were associated with the survival. AMPK levels, tumor grade, and age were independent prognostic factors predicting poor outcomes in patients with astrocytoma. Together, these results indicate that these 4 targets may play a crucial role in the progression and prognosis of human astrocytomas and that AMPK may represent a potential therapeutic target.

Identifying Drug Candidates for COVID-19 with Large-Scale Drug Screening.

Papain-like protease (PLpro) is critical to COVID-19 infection. Therefore, it is a significant target protein for drug development. We virtually screened a 26,193 compound library against the PLpro of SARS-CoV-2 and identified several drug candidates with convincing binding affinities. The three best compounds all had better estimated binding energy than those of the drug candidates proposed in previous studies. By analyzing the docking results for the drug candidates identified in this and previous studies, we demonstrate that the critical interactions between the compounds and PLpro proposed by the computational approaches are consistent with those proposed by the biological experiments. In addition, the predicted binding energies of the compounds in the dataset showed a similar trend as their IC50 values. The predicted ADME and drug-likeness properties also suggested that these identified compounds can be used for COVID-19 treatment.

Locating the hydrogen atoms in endohedral clusterfullerenes by density functional theory.

There is a unique type of endohedral clusterfullerene containing a hydrogen atom inside the carbon cage (hydrogen-containing clusterfullerenes, HCFs). Unfortunately, the precise positions of the H atoms cannot be determined by powerful single-crystal X-ray diffraction, and thus, the reported internal cluster structures of HCFs are ambiguous. In this study, HCFs were investigated using density functional theory calculations. Various internal cluster structures were obtained for Sc4CNH@Ih(7)-C80 and then carefully inspected to summarize all the favorable H locations in the HCFs. Encouragingly, following these structural characteristics, a new Sc4C2H@Ih(7)-C80 isomer with a µ3-H coordination to three Sc atoms was found to be 12.6 kcal mol-1 more stable than a previously reported isomer. It also holds a much larger SOMO-LUMO gap energy (3.57 vs. 2.36 eV). Its increased stability was further understood by the formation of multicenter bonds (three-center one-electron, three-center two-electron, and even four-center two-electron bonds) and electron density topology analyses. The changed H position may lead to rather different electronic structures, bonding states, and relative stability, indicating its critical role in HCFs. The simulated infrared and Raman spectra based on the new structure also agree fairly well with the experimental observations. Our work not only successfully locates the unpredictable H atom inside HCFs but also demonstrates a practical strategy to quickly determine the internal cluster configurations for more complex clusterfullerenes.

Exploring and engineering PAM-diverse Streptococci Cas9 for PAM-directed bifunctional and titratable gene control in bacteria.

The RNA-guided Cas9s serve as powerful tools for programmable gene editing and regulation; their targeting scopes and efficacies, however, are always constrained by the PAM sequence stringency. Most Streptococci Cas9s, including the prototype SpCas9 from S. pyogenes, specifically recognize a canonical NGG PAM via a conserved RxR PAM-binding motif within the PAM-interaction (PI) domain. Here, SpCas9-based mining unveils three distinct and rarely presented PAM-binding motifs (QxxxR, QxQ and RxQ) among Streptococci Cas9 orthologs. With the catalytically-dead QxxxR-containing SedCas9 from S. equinus, we dissect its NAG PAM specificity and elucidate its underlying recognition mechanism via computational prediction and mutagenesis analysis. Replacing the SedCas9 PI domain with alternate PAM-binding motifs rewires its PAM specificity to NGG or NAA. Moreover, a semi-rational design with minimal mutation creates a SedCas9-NQ variant showing robust activity towards expanded NNG and NAA PAMs, based upon which we engineered a compact ω-SedCas9-NQ transcriptional regulator for PAM-directed bifunctional and titratable gene control. The ω-SedCas9-NQ mediated metabolic reprogramming of endogenous genes in Escherichia coli affords a 2.6-fold increase of 4-hydroxycoumarin production. This work reveals new Cas9 scaffolds with distinct PAM-binding motifs for PAM relaxation and creates a new PAM-diverse Cas9 variant for versatile gene control in bacteria.

Correlation of MTAP immunohistochemical deficiency with CDKN2A homozygous deletion and clinicopathological features in pleomorphic xanthoastrocytoma.

Pleomorphic xanthoastrocytoma (PXA) is a rare tumor ranging from World Health Organization (WHO) grades 2-3 and can potentially recur and metastasize throughout the central nervous system (CNS). Cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) deletion is a frequent genomic alteration of PXA. Methylthioadenosine phosphorylase (MTAP) immunohistochemistry is a promising surrogate marker for CDKN2A homozygous deletion in different cancers but has not been examined in PXA. Therefore, we performed CDKN2A fluorescence in situ hybridization and MTAP immunohistochemistry on specimens from 23 patients with CNS WHO grades 2 (n = 10) and 3 (n = 13) PXAs, including specimens from primary and recurrent tumors, and determined whether MTAP immunohistochemistry correlated with CDKN2A homozygous deletion and clinicopathological features. CDKN2A homozygous deletion was detected in 30% (3/10) and 76.9% (10/13) of CNS WHO grades 2 and 3 PXAs, respectively. In addition, MTAP loss was inconsistent with CDKN2A homozygous deletion (sensitivity = 86.7%, specificity = 100%). Furthermore, CDKN2A homozygous deletion was correlated with WHO grade (p = 0.026) and the Ki-67 labeling index (p = 0.037). Therefore, MTAP immunostaining can be a suitable surrogate marker for CDKN2A homozygous deletions in PXAs, and CDKN2A homozygous deletions may be an important prognostic factor for PXAs.

Application of second-generation sequencing in congenital pulmonary airway malformations.

To investigate the differential expression of genes in whole transcripts of congenital pulmonary airway malformation (CPAM) using second-generation sequencing (also known as next-generation sequencing, NGS) technology. Children with CPAM were strictly screened after setting the criteria, and grouped by taking CPAM parietal tissue and CPAM lesion tissue respectively, and RNA-Seq libraries were established separately using second-generation sequencing technology, followed by differential expression analysis and GO (gene ontology) functional enrichment analysis, KEGG (Kyoto encyclopedia of genes and genomes, a database) pathway analysis and GSEA (Gene Set Enrichment Analysis) analysis. Five cases were screened from 36 children with CPAM, and high-throughput sequencing was performed to obtain 10 whole transcripts of samples with acceptable sequence quality and balanced gene coverage. One aberrantly expressed sample (3b) was found by analysis of principal components, which was excluded and then subjected to differential expression analysis, and 860 up-regulated genes and 203 down-regulated genes. GO functional enrichment analysis of differentially expressed genes demonstrates the functional class and cellular localization of target genes. The whole transcript of CPAM shows obvious gene up and down-regulation, differentially expressed genes are located in specific cells and belong to different functional categories, and NGS can provide an effective means to study the transcriptional regulation of CPAM from the overall transcriptional level.

Clinical Image Feature Analysis and Diagnostic Efficacy Evaluation of Pulmonary Ultrasound in the Diagnosis of Congenital Pulmonary Airway Malformations in Children: Based on a Single Center, Retrospective Cohort Study.

Objective: A single center, retrospective cohort study was conducted to analyze the clinical image features and diagnostic efficiency of pulmonary ultrasound in the diagnosis of congenital pulmonary airway malformations (CPAMs) in children. Methods: The starting and ending time of this study is from May 2019 to December 2021. This study included 200 children with CPAM diagnosed by prenatal ultrasound and postpartum CT imaging (aged from 1 hour to 3 years), including 103 males and 97 females. All of them were diagnosed by fetal ultrasound and were examined by chest X-ray (CXR), chest CT, and lung ultrasound (LUS). The clinical image characteristics and diagnostic efficiency of CXR, chest CT, and LUS in the diagnosis of CPAM in children were analyzed. Results: 200 lesions were limited to single lung, and the most common were right lower lobe, right lower lobe in 80 cases (40.0%), left lower lobe in 60 cases (30.0%), right upper lobe in 30 cases (15.0%), left upper lobe in 20 cases (10.0%), and right middle lobe in 10 cases (5.0%). Among the 200 cases of preoperative CT examination, 196 cases (98.00%) showed lesions and confirmed diagnosis, and 4 cases were missed. Chest X-ray showed multiple focal circular low-density shadow in the right lung, and the heart shadow and mediastinum moved slightly to the left. CXR showed multiple cystic transparent shadows in the left lower lung and slightly to the right of the mediastinum and heart. CXR showed multiple balloon cavities of different sizes in the right lung field, and the mediastinum and heart shadow shifted to the left. The direct signs of LUS (including single or multiple cystic lesions) were not significantly different from those of CXR, but the indirect signs were significantly higher than those of CXR. Conclusion: The most common CT findings of CPAM in children are cystic lesions, especially polycystic lesions, while LUS images of CPAM in children are various. LUS is a noninvasive and nonradiological examination method, which is easy to operate and repeat. LUS can be used for preliminary qualitative screening of CPAM in children, and the diagnostic value of indirect signs of LUS is better than that of CXR.

Glioneuronal tumor with neuropil-like islands in the spinal cord: A case report and literature review.

RATIONALE: Glioneuronal tumor with neuropil-like islands (GTNI) is a distinctive neoplasm located in the cerebrum. Moreover, spinal GTNI is extremely rare. Herein, we present a case of spinal GTNI and review the related literature. PATIENT CONCERNS: A 38-year-old Chinese woman presented to our hospital with a 6-month history of neck pain and a 1-month history of dizziness. DIAGNOSES: Magnetic resonance imaging revealed a large intramedullary mass spanning the length of the spinal cord from C1 to C4. Microscopic and immunohistochemical examinations of the tumor tissue revealed findings typical of GTNI. INTERVENTIONS: The patient underwent C1 to C4 intraspinal gross tumor resection. OUTCOMES: Follow-up results showed that the patient had no recurrence 6 months after tumor resection. LESSONS: GTNI in the spinal cord is a highly rare neoplasm with poor prognosis. Therefore, clinicians and pathologists should differentiate GTNI from other benign glioneuronal tumors, and long-term follow-up of patients with spinal GTNI is necessary.

Encapsulating Cas9 into extracellular vesicles by protein myristoylation.

CRISPR/Cas9 genome editing is a very promising avenue for the treatment of a variety of genetic diseases. However, it is still very challenging to encapsulate CRISPR/Cas9 machinery for delivery. Protein N-myristoylation is an irreversible co/post-translational modification that results in the covalent attachment of the myristoyl-group to the N-terminus of a target protein. It serves as an anchor for a protein to associate with the cell membrane and determines its intracellular trafficking and activity. Extracellular vesicles (EVs) are secreted vesicles that mediate cell-cell communication. In this study, we demonstrate that myristoylated proteins were preferentially encapsulated into EVs. The octapeptide derived from the leading sequence of the N-terminus of Src kinase was a favourable substrate for N-myristoyltransferase 1, the enzyme that catalyzes myristoylation. The fusion of the octapeptide onto the N-terminus of Cas9 promoted the myristoylation and encapsulation of Cas9 into EVs. Encapsulation of Cas9 and sgRNA-eGFP inside EVs was confirmed using protease digestion assays. Additionally, to increase the transfection potential, VSV-G was introduced into the EVs. The encapsulated Cas9 in EVs accounted for 0.7% of total EV protein. Importantly, the EVs coated with VSV-G encapsulating Cas9/sgRNA-eGFP showed up to 42% eGFP knock out efficiency with limited off-target effects in recipient cells. Our study provides a novel approach to encapsulate CRISPR/Cas9 protein and sgRNA into EVs. This strategy may open an effective avenue to utilize EVs as vehicles to deliver CRISPR/Cas9 for genome-editing-based gene therapy.

Polyphenols as Potential Inhibitors of SARS-CoV-2 RNA Dependent RNA Polymerase (RdRp).

An increasing number of studies have demonstrated the antiviral nature of polyphenols, and many polyphenols have been proposed to inhibit SARS-CoV or SARS-CoV-2. Our previous study revealed the inhibitory mechanisms of polyphenols against DNA polymerase α and HIV reverse transcriptase to show that polyphenols can block DNA elongation by competing with the incoming NTPs. Here we applied computational approaches to examine if some polyphenols can also inhibit RNA polymerase (RdRp) in SARS-CoV-2, and we identified some better candidates than remdesivir, the FDA-approved drug against RdRp, in terms of estimated binding affinities. The proposed compounds will be further examined to develop new treatments for COVID-19.

Engineering a PAM-flexible SpdCas9 variant as a universal gene repressor.

The RNA-guided CRISPR-associated Cas9 proteins have been widely applied in programmable genome recombination, base editing or gene regulation in both prokaryotes and eukaryotes. SpCas9 from Streptococcus pyogenes is the most extensively engineered Cas9 with robust and manifold functionalities. However, one inherent limitation of SpCas9 is its stringent 5'-NGG-3' PAM requirement that significantly restricts its DNA target range. Here, to repurpose SpCas9 as a universal gene repressor, we generate and screen variants of the deactivated SpCas9 (SpdCas9) with relaxed 5'-CAT-3' PAM compatibility that can bind to the start codon ATG of almost any gene. Stepwise structure-guided mutations of the PAM-interacting residues and auxiliary PAM-proximal residues of the SpdNG (5'-NG-3' PAM) create a PAM-flexible variant SpdNG-LWQT that preferentially accommodates 5'-NRN-3' PAMs. SpdNG-LWQT is demonstrated to be effective in gene repression with the advantage of customizable sgRNA design in both Escherichia coli and Saccharomyces cerevisiae. This work validates the feasibility of purposeful PAM expansion of Cas9 towards signature PAMs and establishes a universal SpdCas9-based gene repressor.

Polyphenols as alternative treatments of COVID-19.

Although scientists around the world have put lots of effort into the development of new treatments for COVID-19 since the outbreak, no drugs except Veklury (remdesivir) have been approved by FDA. There is an urgent need to discover some alternative antiviral treatment for COVID-19. Because polyphenols have been shown to possess antiviral activities, here we conducted a large-scale virtual screening for more than 400 polyphenols. Several lead compounds such as Petunidin 3-O-(6″-p-coumaroyl-glucoside) were identified to have promising binding affinities and convincing binding mechanisms. Analyzing the docking results and ADME properties sheds light on the potential efficacy of the top-ranked drug candidates and pinpoints the key residues on the target proteins for the future of drug development.

Doxorubicin-loaded nanoparticle coated with endothelial cells-derived exosomes for immunogenic chemotherapy of glioblastoma.

Treatments of glioblastoma (GBM) have not been very effective, largely due to the inefficiency of drugs in penetrating the blood brain barrier (BBB). In this study, we investigated the potential of exosome-coated doxorubicin (DOX)-loaded nanoparticles (ENPDOX) in BBB penetration, inducing immunogenic cell death (ICD) and promoting survival of GBM-bearing mice. DOX-loaded nanoparticles (NPDOX) were coated with exosomes prepared from mouse brain endothelial bEnd.3 cells. ENPDOX cellular uptake was examined. Penetration of ENPDOX through the BBB was tested in an in vitro transwell system and a GBM mouse model. The effects of ENPDOX in inducing apoptosis and ICD were assessed. Finally, the efficacy of ENPDOX in the treatment of GBM-bearing mice was assessed. ENPDOX was taken up by bEnd.3 cells and could penetrate the BBB both in vitro and in vivo. In vitro, ENDDOX induced apoptosis and ICD of glioma GL261 cells. Systemic administration of ENPDOX resulted in maturation of dendritic cells, activation of cytotoxic cells, altered production of cytokines, suppressed proliferation and increased apoptosis of GBM cells in vivo and prolonged survival of GBM-bearing mice. Our findings indicate that ENPDOX may be a potent therapeutic strategy for GBM which warrants further investigation in clinical application.

Misdiagnosis of asymptomatic intrathyroidal pyriform sinus fistula: a case report.

Pyriform sinus fistula is uncommon and easily misdiagnosed. Most reported cases occur in children and are associated with either acute suppurative thyroiditis or deep neck infection. Asymptomatic pyriform sinus fistula is difficult to diagnose because it can manifest as an incidental thyroid nodule with highly suspicious malignant features on ultrasonography. The patient was a 41-year-old man with asymptomatic thyroid nodules incidentally detected on ultrasonography. Surgery was performed under the suspicion of thyroid cancer. Pathology findings revealed multiple cystic walls lined by ciliated columnar cells with stratified squamous epithelial cysts in a background of inflammatory and lymphoid cells. Barium swallow examination performed 2 weeks later revealed a sinus tract measuring 1.8 cm that arose from the apex of the left pyriform sinus. The diagnosis and management of pyriform sinus anomalies are challenging. The majority of physicians, including some otolaryngologists, lack an understanding of the disease, which should be considered one of the important differential diagnoses of neck masses. Barium swallow examination, ultrasonography, computed tomography, and laryngoscopy are useful to diagnose this condition.

Pharmacological inhibition of noncanonical EED-EZH2 signaling overcomes chemoresistance in prostate cancer.

Rationale: Chemoresistance is a major obstacle in prostate cancer (PCa) treatment. We sought to understand the underlying mechanism of PCa chemoresistance and discover new treatments to overcome docetaxel resistance. Methods: We developed a novel phenotypic screening platform for the discovery of specific inhibitors of chemoresistant PCa cells. The mechanism of action of the lead compound was investigated using computational, molecular and cellular approaches. The in vivo toxicity and efficacy of the lead compound were evaluated in clinically-relevant animal models. Results: We identified LG1980 as a lead compound that demonstrates high selectivity and potency against chemoresistant PCa cells. Mechanistically, LG1980 binds embryonic ectoderm development (EED), disrupts the interaction between EED and enhancer of zeste homolog 2 (EZH2), thereby inducing the protein degradation of EZH2 and inhibiting the phosphorylation and activity of EZH2. Consequently, LG1980 targets a survival signaling cascade consisting of signal transducer and activator of transcription 3 (Stat3), S-phase kinase-associated protein 2 (SKP2), ATP binding cassette B 1 (ABCB1) and survivin. As a lead compound, LG1980 is well tolerated in mice and effectively suppresses the in vivo growth of chemoresistant PCa and synergistically enhances the efficacy of docetaxel in xenograft models. Conclusions: These results indicate that pharmacological inhibition of EED-EZH2 interaction is a novel strategy for the treatment of chemoresistant PCa. LG1980 and its analogues have the potential to be integrated into standard of care to improve clinical outcomes in PCa patients.

Clinical implications of mismatch repair deficiency screening in patients with mixed neuroendocrine non-neuroendocrine neoplasms (MiNEN).

INTRODUCTION: Mixed neuroendocrine non-neuroendocrine neoplasms (MiNEN) are rare tumors, mainly encountered in the gastroenteropancreatic tract. Based on the limited available data, MiNEN is usually a highly aggressive neoplasm combining a high-grade neuroendocrine and a non-neuroendocrine component, associated with a poor prognostic outlook. Deficient DNA mismatch repair (MMR) results in microsatellite instability, which is a useful screening marker for identifying patients with Lynch syndrome and a prognostic factor for chemotherapeutic interventions. Little information on MMR status in MiNEN is available in published studies. Therefore, the purpose of this study was to explore the status and putative role of MMR on MiNEN. METHODS: We investigated the MMR status in 44 cases and characterized their clinicopathological features and prognoses. Immunohistochemistry was performed for four mismatch repair proteins (MLH1, MSH2, MSH6, and PMS2). RESULTS: Mean age at diagnosis was 61 years, and 75% of the patients were male. Lymph node metastases were observed in 14 (35.9%) patients. The most common tumor localizations were gastric (28 patients, 63.6%). Lack of immunohistochemical expression of MMR proteins was shown in 38.6% of cases. The common deletion rates of one or more proteins were 29.4% (5/17) for MLH1/PMS2 and 23.5% (4/17) for MLH1. Correlation between clinicopathological parameters showed that MMR deficiency was significantly associated with early TNM stage and better prognoses in patients with MiNEN. CONCLUSION: MiNENs showed frequent losses of MMR protein expression, which contributes to the knowledge of the pathological and clinical aspects of MiNEN tumors.