The inhibition of Beclin1-dependent autophagy sensitizes PTC cells to ABT737-induced death.

ABT737 is used as a specific BCL2 inhibitor, which can treat papillary thyroid carcinoma (PTC). However, the effect of ABT737 on PTC cell apoptosis is limited. Moreover, BCL2 inhibition causes the activation of Beclin1-dependent autophagy. Our study aimed to explore the effects of autophagy and Beclin1 on ABT737 efficacy in PTC. The experimental data showed that ABT737 synchronously enhanced autophagic activity and apoptosis level in PTC cells. ABT737 also promoted the dissociation of BCL2-Beclin1 and BCL2-Bax complexes. Autophagy inhibitors, Bafilomycin A1 and 3-MA, enhanced the inhibitory effect of ABT737 on the survival and function in PTC cells. Consistently, autophagy inhibition with Beclin1 pharmacological inhibitor (spautin-1) also enhanced the efficacy of ABT737. Additionally, ABT737 at low-dose promoted LC3 conversion in PTC cells, and did not affect PTC cell apoptosis and survival. However, The efficacy of low-dose of ABT737 in PTC cell apoptosis and survival was displayed with the addition of Bafilomycin A1, 3-MA or spautin-1. In conclusion, the limited role of ABT737 in PTC cell apoptosis is attributed to its promoting effect on Beclin1-dependent autophagy. Therefore, autophagy inhibition based on Beclin1 downregulation can enhance the sensitivity of PTC cells to ABT737-induced death.

Pseudoginsenoside GQ mitigates chronic intermittent hypoxia-induced cognitive damage by modulating microglia polarization.

Obstructive sleep apnea (OSA), a state of sleep disruption, is characterized by recurrent apnea, chronic intermittent hypoxia (CIH) and hypercapnia. Previous studies have showed that CIH-induced neuroinflammatory plays a crucial role in cognitive deficits. Pseudoginsenoside GQ (PGQ) is a new oxytetracycline-type saponin formed by the oxidation and cyclization of the 20(S) Rg3 side chain. Rg3 has been found to afford anti-inflammatory effects, while whether PGQ plays a role of anti-neuroinflammatory remains unclear. The purpose of this study was to investigate whether PGQ attenuates CIH-induced neuroinflammatory and cognitive impairment and the possible mechanism it involves. We found that PGQ significantly ameliorated CIH-induced spatial learning deficits, and inhibited microglial activation, pro-inflammatory cytokine release, and neuronal apoptosis in the hippocampus of CIH mice. In addition, PGQ pretreatment promoted microglial M1 to M2 phenotypic transition in IH-induced BV-2 microglial, as well as indirectly inhibited IH-induced neuronal injury via modulation of microglia polarization. Furthermore, we noted that activation of HMGB1/TLR4/NF-κB signaling pathway induced by IH was inhibited by PGQ. Molecular docking results revealed that PGQ could bind to the active sites of HMGB1 and TLR4. Taken together, this work supports that PGQ inhibits M1 microglial polarization via the HMGB1/TLR4/NF-κB signaling pathway, and indirectly exerts neuroprotective effects, suggesting that PGQ may be a potential therapeutic strategy for cognitive impairment accompanied OSA.

pan-MHC and cross-Species Prediction of T Cell Receptor-Antigen Binding.

Profiling the binding of T cell receptors (TCRs) of T cells to antigenic peptides presented by MHC proteins is one of the most important unsolved problems in modern immunology. Experimental methods to probe TCR-antigen interactions are slow, labor-intensive, costly, and yield moderate throughput. To address this problem, we developed pMTnet-omni, an Artificial Intelligence (AI) system based on hybrid protein sequence and structure information, to predict the pairing of TCRs of αß T cells with peptide-MHC complexes (pMHCs). pMTnet-omni is capable of handling peptides presented by both class I and II pMHCs, and capable of handling both human and mouse TCR-pMHC pairs, through information sharing enabled this hybrid design. pMTnet-omni achieves a high overall Area Under the Curve of Receiver Operator Characteristics (AUROC) of 0.888, which surpasses competing tools by a large margin. We showed that pMTnet-omni can distinguish binding affinity of TCRs with similar sequences. Across a range of datasets from various biological contexts, pMTnet-omni characterized the longitudinal evolution and spatial heterogeneity of TCR-pMHC interactions and their functional impact. We successfully developed a biomarker based on pMTnet-omni for predicting immune-related adverse events of immune checkpoint inhibitor (ICI) treatment in a cohort of 57 ICI-treated patients. pMTnet-omni represents a major advance towards developing a clinically usable AI system for TCR-pMHC pairing prediction that can aid the design and implementation of TCR-based immunotherapeutics.

PHACTR1 promotes the mobility of papillary thyroid carcinoma cells by inducing F-actin formation.

Papillary thyroid carcinoma (PTC) limits effective biomarkers for predicting prognosis and targeted therapy. Phosphatase and actin regulator 1 (PHACTR1) is a mobility-promoting molecule due to its regulation on F-actin formation, which is valuable for the investigation of PTC. Our study aimed to investigate the relationship between PHACTR1 and PTC carcinogenesis, especially mobility. Our results displayed that PHACTR1 expression was elevated in metastatic or larger PTC tissues. In addition, PTC cells K1 with more obvious mobility had higher PHACTR1 expression whereas weakly mobile cells TPC-1 was contrary. Moreover, PHACTR1 silencing inhibited the invasion, migration and tumorigenicity of K1 cells, while PHACTR1 overexpression promoted the invasion, migration and tumorigenicity in TPC-1 cells. Furthermore, PHACTR1 overexpression increased the fluorescent intensity of F-actin in TPC-1 cells. Importantly, the enhanced invasion and migration in TPC-1 cells caused by PHACTR1 overexpression were significantly reversed by the disruption of F-actin assembly with swinholide A. In conclusion, PHACTR1 can promote the mobility of PTC cells, which results in the carcinogenesis of PTC. PHACTR1-regulated F-actin formation determines the mobility of PTC cells. Therefore, PHACTR1 can function as a potential biomarker for predicting prognosis and targeting therapy in PTC.

Abnormal activation of NF-κB and MAPK signaling pathways affect osimertinib resistance and influence the recruitment of myeloid-derived suppressor cells to shape the immunosuppressive tumor immune microenvironment.

BACKGROUND: Osimertinib is the first-line treatment for patients with epidermal growth factor receptor (EGFR) mutations, but the treatment options after drug resistance are limited. Previous studies have suggested that EGFR is in an immunosuppressive tumor immune microenvironment (TIME). However, the evolution of TIME after osimertinib resistance and whether this resistance can be overcome by targeting TIME needs to be further investigated. METHODS: The remodeling process and mechanism of TIME during the treatment with osimertinib were studied. RESULTS: The proportion of EGFRL858R+T790M mutant tumor immune infiltrating cells was extremely low. Osimertinib treatment transiently triggered inflammatory cells, but several immunosuppressive cells infiltrated after drug resistance and formed a myeloid-derived suppressor cell (MDSC)-enriched TIME. The programmed cell death protein-1 monoclonal antibody was not able to reverse the MDSC-enriched TIME. Further analysis revealed that the activation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways recruited a large number of MDSCs via cytokines. Finally, MDSC secreted high levels of interleukin-10 and arginase-1 and created an immunosuppressive TIME. CONCLUSIONS: Thus, our findings lay the foundation for the evolution of TIME in osimertinib treatment, establish the mechanism of immunosuppressive TIME after osimertinib resistance, and propose potential solutions.

ZEB1 Is Regulated by K811 Acetylation to Promote Stability, NuRD Complex Interactions, EMT, and NSCLC Metastasis.

Epithelial-to-mesenchymal transition results in loss of specialized epithelial cell contacts and acquisition of mesenchymal invasive capacity. The transcription repressor zinc finger E-box-binding homeobox 1 (ZEB1) binds to E-boxes of gene promoter regions to suppress the expression of epithelial genes. ZEB1 has inconsistent molecular weights, which have been attributed to posttranslational modifications (PTM). We performed mass spectrometry and identified K811 acetylation as a novel PTM in ZEB1. To define the role of ZEB1 acetylation in regulating function, we generated ZEB1 acetyl-mimetic (K811Q) and acetyl-deficient (K811R) mutant-expressing non-small cell lung cancer cell lines (NSCLC). We demonstrate that the K811R ZEB1 (125 kDa) has a shorter protein half-life than wild-type (WT) ZEB1 and K811Q ZEB1 (∼225 kDa), suggesting that lack of ZEB1 acetylation in the lower molecular weight form affects protein stability. Further, the acetylated form of ZEB1 recruits the nucleosome remodeling and deacetylase (NuRD) complex to bind the promoter of its target genes mir200c-141 and SEMA3F. RNA-sequencing revealed that WT ZEB1 and K811Q ZEB1 downregulate the expression of epithelial genes to promote lung adenocarcinoma invasion and metastasis, whereas the K811R ZEB1 does not. Our findings establish that the K811 acetylation promotes ZEB1 protein stability, interaction with other protein complexes, and subsequent invasion/metastasis of lung adenocarcinoma via epithelial-to-mesenchymal transition. IMPLICATIONS: The molecular mechanisms by which ZEB1 is regulated by K811 acetylation to promote protein stability, NuRD complex and promoter interactions, and function are relevant to the development of treatment strategies to prevent and treat metastasis in patients with NSCLC.

ZEB1 is regulated by K811 acetylation to promote stability, NuRD complex interactions, EMT, and NSCLC metastasis.

Epithelial-to-mesenchymal transition results in loss of specialized epithelial cell contacts and acquisition of mesenchymal invasive capacity. The transcription repressor zinc finger E-box-binding homeobox 1 (ZEB1) binds to E-boxes of gene promoter regions to suppress the expression of epithelial genes. ZEB1 has inconsistent molecular weights, which have been attributed to post-translational modifications (PTMs). We performed mass spectrometry and identified K811 acetylation as a novel PTM in ZEB1. To define the role of ZEB1 acetylation in regulating function, we generated ZEB1 acetyl-mimetic (K811Q) and acetyl-deficient (K811R) mutant-expressing non-small cell lung cancer cell lines (NSCLC). We demonstrate that the K811R ZEB1 (125 kDa) has a shorter protein half-life than wild-type (WT) ZEB1 and K811Q ZEB1 (&tilde225 kDa), suggesting that lack of ZEB1 acetylation in the lower molecular weight form affects protein stability. Further, the acetylated form of ZEB1 recruits the nucleosome remodeling and deacetylase (NuRD) complex to bind the promoter of its target genes mir200c-141 and SEMA3F. RNA-sequencing revealed that WT ZEB1 and K811Q ZEB1 downregulate the expression of epithelial genes to promote lung adenocarcinoma invasion and metastasis, while the K811R ZEB1 does not. Our findings establish that the K811 acetylation promotes ZEB1 protein stability, interaction with other protein complexes, and subsequent invasion/metastasis of lung adenocarcinoma via epithelial-to-mesenchymal transition. Implications: The molecular mechanisms by which ZEB1 is regulated by K811 acetylation to promote protein stability, NuRD complex and promoter interactions, and function are relevant to the development of treatment strategies to prevent and treat metastasis in NSCLC patients.

Genetic trajectory and clonal evolution of multiple primary lung cancer with lymph node metastasis.

Multiple primary lung cancer (MPLC) with lymph node metastasis (LNM) is a rare phenomenon of multifocal lung cancer. The genomic landscapes of MPLC and the clonal evolution pattern between primary lung lesions and lymph node metastasis haven't been fully illustrated. We performed whole-exome sequencing (WES) on 52 FFPE (Formalin-fixed Paraffin-Embedded) samples from 11 patients diagnosed with MPLC with LNM. Genomic profiling and phylogenetic analysis were conducted to infer the evolutional trajectory within each patient. The top 5 most frequently mutated genes in our study were TTN (76.74%), MUC16 (62.79%), MUC19 (55.81%), FRG1 (46.51%), and NBPF20 (46.51%). For most patients in our study, a substantial of genetic alterations were mutually exclusive among the multiple pulmonary tumors of the same patient, suggesting their heterogenous origins. Individually, the genetic profile of lymph node metastatic lesions overlapped with that of multiple lung cancers in different degrees but are more genetically related to specific pulmonary lesions. SETD2 was a potential metastasis biomarker of MPLC. The mean putative neo-antigen number of the primary tumor (646.5) is higher than that of lymph node metastases (300, p = 0.2416). Primary lung tumors and lymph node metastases are highly heterogenous in immune repertoires. Our findings portrayed the comprehensive genomic landscape of MPLC with LNM. We characterized the genomic heterogeneity among different tumors. We offered novel clues to the clonal evolution between MPLC and their lymphatic metastases, thus advancing the treatment strategies and preventions of MPLC with LNM.

TAT-Beclin 1 represses the carcinogenesis of DUSP4-positive PTC by enhancing autophagy.

BACKGROUND: DUSP4 is a pro-tumorigenic molecule of papillary thyroid carcinoma (PTC). DUSP4 also exists as an autophagic regulator. Moreover, DUSP4, as a negative regulator of MAPK, can prevent Beclin 1 from participating in autophagic response. This study aimed to explore whether TAT-Beclin 1, a recombinant protein of Beclin 1, could inhibit the tumorigenesis of DUSP4-positive PTC by regulating autophagy. METHODS: First, we divided PTC tissues into three groups according to DUSP4 expression levels by immunohistochemical analyses, and evaluated the relationship between autophagic molecules (Beclin 1 and LC3II) and DUSP4 using Western blotting assays. After overexpression of DUSP4 by lentiviral transduction, the in vitro and in vivo roles of TAT-Beclin 1 on DUSP4-overexpressed PTC cells were assessed (including autophagic activity, cell survival and function, and tumor growth). The roles of TAT-Beclin 1 in the survival of DUSP4-silenced PTC cells were also evaluated. RESULTS: Our results showed that the expression levels of autophagic proteins decreased with the increase of DUSP4 expression in PTC tissues. In PTC cells, DUSP4 overexpression-inhibited autophagic activity (including Beclin 1 expression, LC3 conversion rate and LC3-puncta formation) and -promoted cell proliferation and migration were reversed by TAT-Beclin 1 administration. In vivo assays also showed that DUSP4-overexpressed PTC cells had stronger tumorigenic ability and weaker autophagic activity, which was blocked by TAT-Beclin 1 administration. CONCLUSION: TAT-Beclin 1, as an autophagic promoter, could repress the carcinogenesis of DUSP4-positive PTC, which implies that the use of TAT-Beclin 1 for the PTC patients' treatment might be determined according to the DUSP4 level in their tumors.

N6-methylandenosine-related immune genes correlate with prognosis and immune landscapes in gastric cancer.

Objectives: This study aimed to probe into the significance of N6-methyladenosine (m6A)-related immune genes (m6AIGs) in predicting prognoses and immune landscapes of patients with gastric cancer (GC). Methods: The clinical data and transcriptomic matrix of GC patients were acquired from The Cancer Genome Atlas database. The clinically meaningful m6AIGs were acquired by univariate Cox regression analysis. GC patients were stratified into different clusters via consensus clustering analysis and different risk subgroups via m6AIGs prognostic signature. The clinicopathological features and tumor microenvironment (TME) in the different clusters and different risk subgroups were explored. The predictive performance was evaluated using the KM method, ROC curves, and univariate and multivariate regression analyses. Moreover, we fabricated a nomogram based on risk scores and clinical risk characteristics. Biological functional analysis was performed based on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways. The connectivity map was used to screen out potential small molecule drugs for GC patients. Results: A total of 14 prognostic m6AIGs and two clusters based on 14 prognostic m6AIGs were identified. A prognostic signature based on 4 m6AIGs and a nomogram based on independent prognostic factors was constructed and validated. Different clusters and different risk subgroups were significantly correlated with TME scores, the distribution of immune cells, and the expression of immune checkpoint genes. Some malignant and immune biological processes and pathways were correlated with the patients with poor prognosis. Ten small molecular drugs with potential therapeutic effect were screened out. Conclusions: This study revealed the prognostic role and significant values of m6AIGs in GC, which enhanced the understanding of m6AIGs and paved the way for developing predictive biomarkers and therapeutic targets for GC.

Single-cell transcriptomic profiling reveals the tumor heterogeneity of small-cell lung cancer.

Small-cell lung cancer (SCLC) is the most aggressive and lethal subtype of lung cancer, for which, better understandings of its biology are urgently needed. Single-cell sequencing technologies provide an opportunity to profile individual cells within the tumor microenvironment (TME) and investigate their roles in tumorigenic processes. Here, we performed high-precision single-cell transcriptomic analysis of ~5000 individual cells from primary tumors (PTs) and matched normal adjacent tissues (NATs) from 11 SCLC patients, including one patient with both PT and relapsed tumor (RT). The comparison revealed an immunosuppressive landscape of human SCLC. Malignant cells in SCLC tumors exhibited diverse states mainly related to the cell cycle, immune, and hypoxic properties. Our data also revealed the intratumor heterogeneity (ITH) of key transcription factors (TFs) in SCLC and related gene expression patterns and functions. The non-neuroendocrine (non-NE) tumors were correlated with increased inflammatory gene signatures and immune cell infiltrates in SCLC, which contributed to better responses to immune checkpoint inhibitors. These findings indicate a significant heterogeneity of human SCLC, and intensive crosstalk between cancer cells and the TME at single-cell resolution, and thus, set the stage for a better understanding of the biology of SCLC as well as for developing new therapeutics for SCLC.

Molecular profiling of human non-small cell lung cancer by single-cell RNA-seq.

BACKGROUND: Lung cancer, one of the most common malignant tumors, exhibits high inter- and intra-tumor heterogeneity which contributes significantly to treatment resistance and failure. Single-cell RNA sequencing (scRNA-seq) has been widely used to dissect the cellular composition and characterize the molecular properties of cancer cells and their tumor microenvironment in lung cancer. However, the transcriptomic heterogeneity among various cancer cells in non-small cell lung cancer (NSCLC) warrants further illustration. METHODS: To comprehensively analyze the molecular heterogeneity of NSCLC, we performed high-precision single-cell RNA-seq analyses on 7364 individual cells from tumor tissues and matched normal tissues from 19 primary lung cancer patients and 1 pulmonary chondroid hamartoma patient. RESULTS: In 6 of 16 patients sequenced, we identified a significant proportion of cancer cells simultaneously expressing classical marker genes for two or even three histologic subtypes of NSCLC-adenocarcinoma (ADC), squamous cell carcinoma (SCC), and neuroendocrine tumor (NET) in the same individual cell, which we defined as mixed-lineage tumor cells; this was verified by both co-immunostaining and RNA in situ hybridization. These data suggest that mixed-lineage tumor cells are highly plastic with mixed features of different types of NSCLC. Both copy number variation (CNV) patterns and mitochondrial mutations clearly showed that the mixed-lineage and single-lineage tumor cells from the same patient had common tumor ancestors rather than different origins. Moreover, we revealed that patients with high mixed-lineage features of different cancer subtypes had worse survival than patients with low mixed-lineage features, indicating that mixed-lineage tumor features were associated with poorer prognosis. In addition, gene signatures specific to mixed-lineage tumor cells were identified, including AKR1B1. Gene knockdown and small molecule inhibition of AKR1B1 can significantly decrease cell proliferation and promote cell apoptosis, suggesting that AKR1B1 plays an important role in tumorigenesis and can serve as a candidate target for tumor therapy of NSCLC patients with mixed-lineage tumor features. CONCLUSIONS: In summary, our work provides novel insights into the tumor heterogeneity of NSCLC in terms of the identification of prevalent mixed-lineage subpopulations of cancer cells with combined signatures of SCC, ADC, and NET and offers clues for potential treatment strategies in these patients.

PSC subtyping based on TTF-1 and p40 expression reveals distinct molecular characteristics and therapeutic strategies.

Pulmonary sarcomatoid carcinoma (PSC) is a unique form of poorly differentiated nonsmall cell lung cancer (NSCLC) and is notorious for its highly malignant nature and dismal prognosis. To introduce effective treatment for PSC patients, precise subtyping of PSC is demanding. In our study, TTF-1 and P40 immunohistochemistry (IHC) staining were applied to 56 PSC patients with multiomics data. According to IHC results, we categorized these patients into three subgroups and profiled their molecular contexture using bioinformatic skills. IHC results classified these patients into three subgroups: TTF-1 positive subgroup (n = 27), P40 positive subgroup (n = 15) and double-negative subgroup (n = 14). Spindle cell samples accounted for 35.71% (5/14) of double-negative patients, higher than others (P = .034). The three subgroups were heterogeneous in the genomic alteration spectrum, showing significant differences in the RTK/RAS pathway (P = .004) and the cell cycle pathway (P = .030). The methylation profile of the double-negative subgroup was between the other two subgroups. In similarity analysis, the TTF-1 and p40 subgroups were closely related to LUAD and LUSC, respectively. The TTF-1 positive subgroup had the highest leukocyte fraction (LF) among several cancer types, and the tumor mutation burden (TMB) of the p40 positive subgroup ranked third in the TMB list, suggesting the applicability of immunotherapy for PSC. The study established a new subtyping method of PSC based on IHC results and reveals three subgroups with distinct molecular features, providing evidence for refined stratification in the treatment of PSC.

Analysis of the Diagnostic Effect of EUS-RTE on Giant Cystic Tumours of the Oesophagus Based on Cluster Analysis.

This paper presents an in-depth analysis and study of the diagnostic effectiveness of EUS-RTE in giant cystic tumours of the oesophagus utilizing cluster analysis. A new form of interval data expression was designed based on the cluster analysis algorithm, as well as a new way of updating the cluster radius and cluster centre. Feature triads are defined, eliminating the need to access all historical data at the time of update. It also prevents the case of overfusion of clusters and outputting only one cluster. If there exist a very low number of clusters, the newly merged clusters are reclustered according to the density clustering method for the internal data objects based on the cluster segmentation so that the data objects in the same cluster have a high similarity as possible. All accumulated electronic files of oesophageal cancer cases were collected and comprehensively organized, and all clinical data of 129 eligible cases with a total of 356 consultations were screened in strict accordance with inclusion and exclusion criteria. A database of oesophageal cancer cases was established using Visual FoxPro software, and frequency distribution, cluster analysis, association rule, and chi-square test were used to focus on mining the association between symptoms, disease mechanisms, prescriptions, and medications. The results were analysed and summarized. Overall, the therapeutic efficacy and safety of the three groups of treatment modalities for gastric mesenchymal tumours were positive, and the preoperative endoscopic treatment modalities should be selected based on the EUS-RTE characteristics of the tumour, the site, and the operator's skill level in a comprehensive manner.

Cold and heterogeneous T cell repertoire is associated with copy number aberrations and loss of immune genes in small-cell lung cancer.

Small-cell lung cancer (SCLC) is speculated to harbor complex genomic intratumor heterogeneity (ITH) associated with high recurrence rate and suboptimal response to immunotherapy. Here, using multi-region whole exome/T cell receptor (TCR) sequencing as well as immunohistochemistry, we reveal a rather homogeneous mutational landscape but extremely cold and heterogeneous TCR repertoire in limited-stage SCLC tumors (LS-SCLCs). Compared to localized non-small cell lung cancers, LS-SCLCs have similar predicted neoantigen burden and genomic ITH, but significantly colder and more heterogeneous TCR repertoire associated with higher chromosomal copy number aberration (CNA) burden. Furthermore, copy number loss of IFN-γ pathway genes is frequently observed and positively correlates with CNA burden. Higher mutational burden, higher T cell infiltration and positive PD-L1 expression are associated with longer overall survival (OS), while higher CNA burden is associated with shorter OS in patients with LS-SCLC.

Circ_0027599 elevates RUNX1 expression via sponging miR-21-5p on gastric cancer progression.

BACKGROUND: Increasing evidence has shown that circular RNAs (circRNAs) serve as vital regulators in tumour progression. In this study, we focused on the functions of circ_0027599 in gastric cancer (GC) progression. METHODS: The levels of circ_0027599, runt-related transcription factor 1 (RUNX1) mRNA and microRNA-21-5p (miR-21-5p) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) assay. The protein levels of RUNX1, E-Cadherin, vimentin and N-Cadherin were measured by Western blot assay. Cell viability, colony formation, metastasis and cell cycle process were evaluated by Cell Counting Kit-8 (CCK-8) assay, colony formation assay, transwell assay and flow cytometry analysis, respectively. The interaction between circ_0027599 and miR-21-5p and the interaction between miR-21-5p and RUNX1 were verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. The role of circ_0027599 in tumour growth in vivo was investigated by murine xenograft model assay. RESULTS: Circ_0027599 and RUNX1 were downregulated in GC tissues and cells. Circ_0027599 level was associated with the overall survival of GC patients. Circ_0027599 or RUNX1 overexpression inhibited GC cell viability, colony formation, migration, invasion and cell cycle process in vitro. For mechanism analysis, circ_0027599 positively regulated RUNX1 expression via functioning as the sponge for miR-21-5p. RUNX1 inhibition reversed circ_0027599 overexpression mediated malignant behaviours of GC cells. Moreover, circ_0027599 overexpression repressed tumour growth in vivo. CONCLUSION: Circ_0027599 overexpression repressed GC progression via modulation of miR-21-5p/RUNX1 axis, which might illumine a novel therapeutic target for GC.

miR­122­5p suppresses the oncogenesis of PTC by inhibiting DUSP4 expression.

MicroRNAs (miRNAs or miRs) play an important role in regulating the occurrence and development of papillary thyroid carcinoma (PTC). miR­122­5p is widely considered a tumour inhibitor, which has not been fully explored in PTC. Bioinformatics analysis identified dual specificity phosphatase 4 (DUSP4), a tumour promoter gene for PTC, as a downstream target of miR­122­5p. The aim of the present study was to investigate the role and molecular mechanism of miR­122­5p in PTC oncogenesis. In this study, the expression pattern of miR­122­5p in PTC cancer tissues and PTC cell lines was investigated via reverse transcription­quantitative PCR. Furthermore, the roles of miR­122­5p in PTC were explored using gain­of­function and loss­of­function assays. The results revealed that the expression of miR­122­5p was significantly lower in PTC cancer tissues, especially in cancer tissues with significant invasion or metastasis. Overexpression of miR­122­5p caused by miR­122­5p mimics inhibited the proliferation, invasion, and migration of the PTC cell line K1, while knockdown of miR­122­5p by miR­122­5p inhibitors exhibited the opposite effect. Furthermore, in vivo assays revealed that miR­122­5p overexpression inhibited tumour growth. In addition, miR­122­5p was negatively correlated with DUSP4 expression in PTC cancer tissues. miR­122­5p overexpression inhibited DUSP4 expression in K1 cells, while miR­122­5p downregulation produced the inverse effect. Specifically, a luciferase reporter assay confirmed the binding sites of miR­122­5p on the 3'­UTR of DUSP4, demonstrating the targeting effect of miR­122­5p on DUSP4. miR­122­5p inhibited the oncogenesis of PTC by targeting DUSP4, revealing the potential application value of miR­122­5p in the diagnosis and treatment of PTC.

ERAP2 Is Associated With Immune Infiltration and Predicts Favorable Prognosis in SqCLC.

Background: Immunotherapy has been proven effective among several human cancer types, including Squamous cell lung carcinoma (SqCLC). ERAP2 plays a pivotal role in peptide trimming of many immunological processes. However, the prognostic role of ERAP2 and its relationship with immune cell infiltration in SqCLC remains unclear. Methods: The differential expression of ERAP2 was identified via GEO and TCGA databases. We calculated the impact of ERAP2 on clinical prognosis using the Kaplan-Meier plotter. TIMER was applied to evaluate the abundance of immune cells infiltration and immune markers. SqCLC tissue microarrays containing 190 patients were constructed, and we performed immunohistochemical staining for ERAP2, CD8, CD47, CD68, and PD-L1 to validate our findings in public data. Results: In the GEO SqCLC database, ERAP2 was upregulated in patients with better survival (p=0.001). ERAP2 expression in SqCLC was significantly lower than that of matched normal samples (p<0.05) based on TCGA SqCLC data. Higher expression of ERAP2 was significantly associated with better survival in SqCLC patients from TCGA (p=0.007), KM-plotter (p=0.017), and our tissue microarrays (TMAs) (p=0.026). In univariate and multivariate Cox analysis of SqCLC TMAs, high ERAP2 expression was identified as an independent protective factor for SqCLC patients (Univariate Cox, HR=0.659, range 0.454-0.956, p<0.05. Multivariate Cox, HR=0.578, range 0.385-0.866, p<0.05). In TIMER, ERAP2 was positively correlated with several immune markers (CD274, p=1.27E-04; CD68, p=5.88E-08) and immune infiltrating cells (CD8+ T cell, p=4.09E-03; NK cell, p=1.00E-04). In our cohort, ERAP2 was significantly correlated with CD8+ tumor-infiltrating lymphocytes (TILs) (p=0.0029), and patients with higher ERAP2 expression had a higher percentage of PD-L1 positive patients (p=0.049) and a higher CD8+ TILs level (p=0.036). Conclusions: For the first time, our study demonstrates that higher expression of ERAP2 is tightly associated with the immuno-supportive microenvironment and can predict a favorable prognosis in SqCLC. Meanwhile, ERAP2 may be a promising immunotherapeutic target for patients with SqCLC.

Upfront dose-reduced chemotherapy synergizes with immunotherapy to optimize chemoimmunotherapy in squamous cell lung carcinoma.

BACKGROUND: The survival benefits of combining chemotherapy (at the maximum tolerated dose, MTD) with concurrent immunotherapy, collectively referred to as chemoimmunotherapy, for the treatment of squamous cell lung carcinoma (SQCLC) have been confirmed in recent clinical trials. Nevertheless, optimization of chemoimmunotherapy in order to enhance the efficacy of immune checkpoint inhibitors (ICIs) in SQCLC remains to be explored. METHODS: Cell lines, syngeneic immunocompetent mouse models, and patients' peripheral blood mononuclear cells were used in order to comprehensively explore how to enhance ectopic lymphoid-like structures (ELSs) and upregulate the therapeutic targets of anti-programmed death 1 (PD-1)/anti-PD-1 ligand (PD-L1) monoclonal antibodies (mAbs), thus rendering SQCLC more sensitive to ICIs. In addition, molecular mechanisms underlying optimization were characterized. RESULTS: Low-dose chemotherapy contributed to an enhanced antigen exposure via the phosphatidylinositol 3-kinase/Akt/transcription factor nuclear factor kappa B signaling pathway. Improved antigen uptake and presentation by activated dendritic cells (DCs) was observed, thus invoking specific T cell responses leading to systemic immune responses and immunological memory. In turn, enhanced antitumor ELSs and PD-1/PD-L1 expression was observed in vivo. Moreover, upfront metronomic (low-dose and frequent administration) chemotherapy extended the time window of the immunostimulatory effect and effectively synergized with anti-PD-1/PD-L1 mAbs. A possible mechanism underlying this synergy is the increase of activated type I macrophages, DCs, and cytotoxic CD8+ T cells, as well as the maintenance of intestinal gut microbiota diversity and composition. In contrast, when combining routine MTD chemotherapy with ICIs, the effects appeared to be additive rather than synergistic. CONCLUSIONS: We first attempted to optimize chemoimmunotherapy for SQCLC by investigating different combinatorial modes. Compared with the MTD chemotherapy used in current clinical practice, upfront metronomic chemotherapy performed better with subsequent anti-PD-1/PD-L1 mAb treatment. This combination approach is worth investigating in other types of tumors, followed by translation into the clinic in the future.

Refined Stratification Based on Baseline Concomitant Mutations and Longitudinal Circulating Tumor DNA Monitoring in Advanced EGFR-Mutant Lung Adenocarcinoma Under Gefitinib Treatment.

INTRODUCTION: The optimal treatment for EGFR-mutant lung adenocarcinoma (LUAD) remains challenging because of intratumor heterogeneity. We aimed to explore a refined stratification model based on the integrated analysis of circulating tumor DNA (ctDNA) tracking. METHODS: ctDNA was prospectively collected at baseline and at every 8 weeks in patients with advanced treatment-naive EGFR-mutant LUAD under gefitinib treatment enrolled in a phase 2 trial and analyzed using next-generation sequencing of a 168-gene panel. RESULTS: Three subgroups categorized by baseline comutations-EGFR-sensitizing mutations (59, 32.8%), EGFR-sensitizing mutations with tumor suppressor mutations (97, 53.9%), and EGFR-sensitizing mutations with other driver mutations (24, 13.3%)-exhibited distinct progression-free survival (13.2 [11.3-15.2] versus 9.3 [7.6-10.5] versus 4.0 [2.4-9.3] months) and overall survival (32.0 [29.2-41.5] versus 21.7 [19.3-27.0] versus 15.5 [10.5-33.7] months, respectively), providing evidence for initial stratification. A total of 63.7% of the patients achieved week 8 ctDNA clearance, with significant difference noted among the three subgroups (74.5% versus 64.0% versus 29.4%, respectively, p = 0.004, Fisher's exact test). Patients without week 8 ctDNA clearance had worse progression-free survival (clearance versus nonclearance 11.2 [9.9-13.2] versus 7.4 [5.6-9.6] months, p = 0.016, Cox regression], especially in the second subgroup [5.8 (5.6-11.5) months], suggesting the necessity of adaptive stratification during treatment. During follow-up, 56.0% and 20.8% of the patients eventually harbored p.T790M and non-p.T790M mutations, respectively, with a significant difference in non-p.T790M mutations among the three subgroups (7.5% versus 15.7% versus 80.0%, respectively, p < 0.001, Fisher's exact test), giving clues to postline treatment. CONCLUSIONS: The patients with baseline comutations and ctDNA nonclearance at first visit might require combined therapy because of the limited survival benefit of EGFR tyrosine kinase inhibitor monotherapy. We proposed a refined stratification mode for the whole-course management of EGFR-mutant LUAD.