MAFF confers vulnerability to cisplatin-based and ionizing radiation treatments by modulating ferroptosis and cell cycle progression in lung adenocarcinoma.

AIMS: Lung cancer is the leading cause of cancer mortality and lung adenocarcinoma (LUAD) accounts for more than half of all lung cancer cases. Tumor elimination is mostly hindered by drug resistance and the mechanisms remain to be explored in LUAD. METHODS: CRISPR screens in cell and murine models and single-cell RNA sequencing were conducted, which identified MAF bZIP transcription factor F (MAFF) as a critical factor regulating tumor growth and treatment resistance in LUAD. RNA and ChIP sequencing analyses were performed for transcriptional target expression and specific binding sites of MAFF. Functions of MAFF in inhibiting tumor growth and promoting cisplatin or irradiation efficacy were investigated using cellular and xenograft models. RESULTS: Patients with lung adenocarcinoma and reduced MAFF expression had worse clinical outcomes. MAFF inhibited tumor cell proliferation by regulating the expression of SLC7A11, CDK6, and CDKN2C, promoting ferroptosis and preventing cell cycle progression from G1 to S. MAFF also conferred tumor cells vulnerable to cisplatin-based or ionizing radiation treatments. MAFF reduction was a final event in the acquisition of cisplatin resistance of LUAD cells. The intracellular cAMP/PKA/CREB1 pathway upregulated MAFF in response to cisplatin-based or ionizing radiation treatments. CONCLUSIONS: MAFF suppresses tumor growth, and pharmacological agonists targeting MAFF may improve cisplatin or irradiation therapies for lung adenocarcinoma patients.

Inhibition of PKA/CREB1 pathway confers sensitivity to ferroptosis in non-small cell lung cancer.

Ferroptosis is a type of regulated cell death characterized by iron accumulation and lipid peroxidation. The molecular mechanisms underlying ferroptosis regulation in non-small cell lung cancer (NSCLC) are poorly understood. In this study, we found that protein kinase A (PKA) inhibition enhanced ferroptosis susceptibility in NSCLC cells, as evidenced by reduced cell viability and increased lipid peroxidation. We further identified cAMP-responsive element protein 1 (CREB1), a transcription factor and a substrate of PKA, as a key regulator of ferroptosis. Knockdown of CREB1 sensitized NSCLC cells to ferroptosis inducers (FINs) and abolished the effects of PKA inhibitor and agonist, revealing the pivotal role of CREB1 in ferroptosis regulation. Using a high-throughput screening approach and subsequent validation by chromatin immunoprecipitation (ChIP) and dual-luciferase assays, we discovered that CREB1 transcriptionally activated stearoyl-CoA desaturase (SCD), an enzyme that catalyzes the conversion of saturated fatty acids to monounsaturated fatty acids. SCD conferred ferroptosis resistance by decreasing the availability of polyunsaturated fatty acids for lipid peroxidation, and its overexpression rescued the effect of CREB1 knockdown on ferroptosis in vitro. Besides, CREB1 knockdown suppressed xenograft tumor growth in the presence of Imidazole Ketone Erastin (IKE), a potent FIN, and this effect was reversed by SCD. Finally, we showed that high expression of CREB1 was associated with poor prognosis in NSCLC patients from public datasets and our institution. Collectively, this study illustrates the effect of PKA/CREB1/SCD axis in regulating ferroptosis of NSCLC, targeting this pathway may provide new strategies for treating NSCLC patients.

Cisplatin resistance-related multi-omics differences and the establishment of machine learning models.

OBJECTIVES: Platinum-based chemotherapies are currently the first-line treatment of non-small cell lung cancer. This study will improve our understanding of the causes of resistance to cisplatin, especially in lung adenocarcinoma (LUAD) and provide a reference for therapeutic decisions in clinical practice. METHODS: Cancer Cell Line Encyclopedia (CCLE), The Cancer Genome Atlas (TCGA) and Zhongshan hospital affiliated to Fudan University (zs-cohort) were used to identify the multi-omics differences related to platinum chemotherapy. Cisplatin-resistant mRNA and miRNA models were constructed by Logistic regression, classification and regression tree and C4.5 decision tree classification algorithm with previous feature selection performed via least absolute shrinkage and selection operator (LASSO). qRT-PCR and western-blotting of A549 and H358 cells, as well as single-cell Seq data of tumor samples were applied to verify the tendency of certain genes. RESULTS: 661 cell lines were divided into three groups according to the IC50 value of cisplatin, and the top 1/3 (220) with a small IC50 value were defined as the sensitive group while the last 1/3 (220) were enrolled in the insensitive group. TP53 was the most common mutation in the insensitive group, in contrast to TTN in the sensitive group. 1348 mRNA, 80 miRNA, and 15 metabolites were differentially expressed between 2 groups (P < 0.05). According to the LASSO penalized logistic modeling, 6 of the 1348 mRNAs, FOXA2, BATF3, SIX1, HOXA1, ZBTB38, IRF5, were selected as the associated features with cisplatin resistance and for the contribution of predictive mRNA model (all of adjusted P-values < 0.001). Three of 6 (BATF3, IRF5, ZBTB38) genes were finally verified in cell level and patients in zs-cohort. CONCLUSIONS: Somatic mutations, mRNA expressions, miRNA expressions, metabolites and methylation were related to the resistance of cisplatin. The models we created could help in the prediction of the reaction and prognosis of patients given platinum-based chemotherapies.

Pan-cancer characterization of metabolism-related biomarkers identifies potential therapeutic targets.

BACKGROUND: Generally, cancer cells undergo metabolic reprogramming to adapt to energetic and biosynthetic requirements that support their uncontrolled proliferation. However, the mutual relationship between two critical metabolic pathways, glycolysis and oxidative phosphorylation (OXPHOS), remains poorly defined. METHODS: We developed a "double-score" system to quantify glycolysis and OXPHOS in 9668 patients across 33 tumor types from The Cancer Genome Atlas and classified them into four metabolic subtypes. Multi-omics bioinformatical analyses was conducted to detect metabolism-related molecular features. RESULTS: Compared with patients with low glycolysis and high OXPHOS (LGHO), those with high glycolysis and low OXPHOS (HGLO) were consistently associated with worse prognosis. We identified common dysregulated molecular features between different metabolic subgroups across multiple cancers, including gene, miRNA, transcription factor, methylation, and somatic alteration, as well as investigated their mutual interfering relationships. CONCLUSION: Overall, this work provides a comprehensive atlas of metabolic heterogeneity on a pan-cancer scale and identified several potential drivers of metabolic rewiring, suggesting corresponding prognostic and therapeutic utility.

Knockdown of GTF2E2 inhibits the growth and progression of lung adenocarcinoma via RPS4X in vitro and in vivo.

BACKGROUND: Lung adenocarcinoma (LUAD) is one of the most common malignancies worldwide. However, the molecular mechanism of LUAD tumorigenesis and development remains unclear. The purpose of this study was to comprehensively illustrate the role of GTF2E2 in the growth and progression of LUAD. METHODS AND MATERIALS: We obtained the mRNA expression data from The Cancer Genome Atlas, Gene Expression Omnibus database, and our institution. Systematic bioinformatical analyses were performed to investigate the expression and prognostic value of GTF2E2 in LUAD. The results were validated by immunohistochemistry and qPCR. The effect of knocking down GTF2E2 using two short hairpin RNAs was investigated by in vitro and in vivo assays. Subsequently, shotgun liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analyses were applied to identified potential GTF2E2 interacting proteins, and the downstream molecular mechanisms of GTF2E2-signaling were further explored by a series of cellular functional assays. RESULTS: We found that GTF2E2 expression was significantly increased in LUAD tissue compared with adjacent normal tissue and was negatively associated with patients' overall survival. Besides, we demonstrated that GTF2E2 knockdown inhibited LUAD cell proliferation, migration, invasion, and promote apoptosis in vitro, as well as attenuated tumor growth in vivo. Results from LC-MS/MS suggested that RPS4X might physically interact with GTF2E2 and mediated GTF2E2's regulatory effect on LUAD development through the mTOR pathway. CONCLUSION: Our findings indicate that GTF2E2 promotes LUAD development by activating RPS4X. Therefore, GTF2E2 might serve as a promising biomarker for the diagnosis and prognosis of LUAD patients, thus shedding light on the precise and personalized therapy for LUAD in the future.

Identification of differentially expressed genes in lung adenocarcinoma cells using single-cell RNA sequencing not detected using traditional RNA sequencing and microarray.

Lung adenocarcinoma (LUAD) is the leading cause of cancer-related deaths worldwide. Traditional RNA sequencing data fails to detect the exact cellular and molecular changes in tumor cells as they make up only a small proportion of tumor tissue. 10× genomics single-cell RNA sequencing (10× scRNA-seq) and gene expression data of LUAD patients was obtained from the Department of Thoracic Surgery, Zhongshan Hospital, Fudan University, ArrayExpress, TCGA, and GEO databases. Differentially expressed genes (DEGs) were identified in LUAD and alveolar cells (DEGs-scRNA-cancer_cell), tumor- and normal tissue-derived cells (DEGs-scRNA-sample), and normal and LUAD patients (DEGs-Bulk). Flow cytometry and qRT-PCR were performed to validate the significantly differentially expressed ligand-receptor pairs. We selected 159,219 cells and 594 samples in the scRNA-seq data and traditional RNA sequencing, respectively. A total of 1042 DEGs-scRNA-cancer_cell, 788 DEGs-scRNA-sample, and 2510 DEGs-Bulk were identified in this study. We also identified 57 DEGs that were only detected in DEGs-scRNA-cancer_cell (only-DEGs-scRNA-cancer_cell). To explore the relationship between only-DEGs-scRNA-cancer_cell and survival in LUAD, 14 and 22 only-DEGs-scRNA-cancer_cell, which were closely related with survival in TCGA and GEO cohorts were identified. Functional enrichment analyses showed these DEGs-scRNA-cancer_cells were mainly related to cell proliferation and immunoregulation. Our study detected and compared DEGs at different levels and revealed genes that may regulate tumor development. Our results provide a potential new protocol to determine the contribution of DEGs to cancer progression and to help identify potential therapeutic targets.

Identification of immune-related gene signature predicting survival in the tumor microenvironment of lung adenocarcinoma.

The tumor microenvironment (TME) plays an essential role in the occurrence and progression of malignancy. The potential prognostic TME-related biomarkers of lung adenocarcinoma (LUAD) remained unclear, which were investigated in this research. The RNA-sequencing profiles and corresponding clinical parameters were extracted from TCGA and GEO databases, based on which the stromal and immune scores were calculated through the ESTIMATE algorithm. Overlapping differentially expressed genes between stromal and immune score group were analyzed by the LASSO and Random Forrest algorithms and validated in cases from our center. And a prognostic 8-gene signature was constructed using Cox regression. The infiltration of 22 hematopoietic cell phenotypes was assessed by the CIBERSORT algorithms. We found that female, elder patients, and solid predominant subtype had obviously higher stromal and immune scores. And patients with early stage LUAD received a prominently higher immune score. A high stromal or immune score meant a good prognosis. Subsequently, eight TME-related prognostic genes (ATAD5, CYP4F3, CYP4F12, ESPNL, FXYD2, GPX2, NLGN4Y, and SERPINC1) were identified by both LASSO regression and Radom Forest algorithms. High 8-gene signature group exhibited worse overall survival. Furthermore, B cell naïve, plasma cells, T cell follicular helper, and macrophages M1 were prominently more in high signature group. Nevertheless, fewer T cells CD4 memory resting, monocytes, and dendritic cell resting were identified in the high signature group. The composition of the tumor microenvironment significantly affected the prognosis of LUAD patients. We provided a new strategy for the exploration of prognostic TME-related biomarkers and immunotherapy.

Identification and validation of tumor environment phenotypes in lung adenocarcinoma by integrative genome-scale analysis.

PURPOSE: To comprehensively elucidate the landscape of the tumor environment (TME) of lung adenocarcinoma (LUAD), which has a profound impact on prognosis and response to immunotherapy. METHODS AND MATERIALS: Using a large dataset of LUAD patients from The Cancer Genome Atlas, Gene Expression Omnibus database (GEO), and our institution (n = 1411), we estimated the infiltration pattern of 24 immune cell populations in each sample and systematically correlated the TME phenotypes with genomic traits and clinicopathologic characteristics. RESULTS: The LUAD microenvironment was classified into two distinct TME clusters (A and B), and a random forest classifier model was constructed. TMEcluster A was characterized by sparse distribution of immune cell infiltration, relatively low levels of immunomodulators and slightly higher mutation load. By contrast, enrichment of both cytotoxic T cells and immunosuppressor cells was observed in TMEcluster B. Moreover, several immune-related cytokines or markers including IFN-γ, TNF-ß, and several immune checkpoint molecules such as PD-L1 were also upregulated in TMEcluster B. Multivariable Cox analysis revealed that the TMEcluster was an independent prognostic factor (TMEcluster B vs. A, hazard ratio = 0.68, 95% confidence interval = 0.50-0.91, p = 0.010). These findings were all externally validated in the data from the GEO database and our institution. CONCLUSIONS: Our findings describe a comprehensive landscape of LUAD immune infiltration pattern and integrate several previously proposed biomarkers associated with distinct immunophenotypes, thus shedding light on how tumors interact with immune microenvironment. Our results may guide a more precise immune therapeutic strategy for LUAD patients.

Log odds of positive lymph nodes predicts survival in patients treated with neoadjuvant therapy followed by esophagectomy.

BACKGROUND AND OBJECTIVES: We aimed to evaluate the efficacy of the log odds of positive lymph nodes (LODDS) in survival prediction of patients with esophageal carcinoma receiving neoadjuvant therapy, compared with N descriptor and positive lymph node ratio (LNR). METHODS: Patients with esophageal carcinoma receiving neoadjuvant therapy from 2004 to 2015 were reviewed in Surveillance, Epidemiology, and End Results database. The receiver operating characteristics curve and area under the curve (AUC) were used to compare discriminatory power among N descriptor, LNR, and LODDS. The goodness of fit was measured using the -2 log-likelihood ratio (-2LLR). RESULTS: About 2239 patients with a 22 months median follow-up and a 37.8% 5-year overall survival rate were included. LODDS had the best discriminatory power and goodness of fit (LODDS vs N descriptor, AUC 0.666 vs 0.626, -2LLR 15 680.402 vs 15 746.162; LODDS vs LNR, AUC 0.666 vs 0.635, -2LLR 15 680.402 vs 15 712.379; all P < .001). LODDS was the best for fewer than 15 lymph nodes retrieved (LODDS vs N descriptor, AUC 0.652 vs 0.618, P < .001; LODDS vs LNR, AUC 0.652 vs 0.625, P = .005). The prognosis of patients without metastatic nodes could be discriminated by LODDS. CONCLUSIONS: LODDS could better predict survival of patients with esophageal carcinoma receiving neoadjuvant therapy.

MNT inhibits lung adenocarcinoma ferroptosis and chemosensitivity by suppressing SAT1.

Ferroptosis, a type of iron-dependent non-apoptotic cell death, plays a vital role in both tumor proliferation and resistance to chemotherapy. Here, our study demonstrates that MAX's Next Tango (MNT), by involving itself in the spermidine/spermine N1-acetyltransferase 1 (SAT1)-related ferroptosis pathway, promotes the proliferation of lung adenocarcinoma (LUAD) cells and diminishes their sensitivity to chemotherapy. Initially, an RNA-sequence screen of LUAD cells treated with ferroptosis inducers (FINs) reveals a significant increase in MNT expression, suggesting a potential link between MNT and ferroptosis. Overexpression of MNT in LUAD cells hinders changes associated with ferroptosis. Moreover, the upregulation of MNT promotes cell proliferation and suppresses chemotherapy sensitivity, while the knockdown of MNT has the opposite effect. Through the intersection of ChIP-Seq and ferroptosis-associated gene sets, and validation by qPCR and western blot, SAT1 is identified as a potential target of MNT. Subsequently, we demonstrate that MNT binds to the promoter sequence of SAT1 and suppresses its transcription by ChIP-qPCR and dual luciferase assays. Restoration of SAT1 levels antagonizes the efficacy of MNT to inhibit ferroptosis and chemosensitivity and promote cell growth in vitro as well as in vivo. In the clinical context, MNT expression is elevated in LUAD and is inversely connected with SAT1 expression. High MNT expression is also associated with poor patient survival. Our research reveals that MNT inhibits ferroptosis, and impairing chemotherapy effectiveness of LUAD.

Transcription factor ZNF263 enhances EGFR-targeted therapeutic response and reduces residual disease in lung adenocarcinoma.

EGF receptor (EGFR) tyrosine kinase inhibitors (TKIs) have achieved clinical success in lung adenocarcinoma (LUAD). However, tumors often show profound but transient initial response and then gain resistance. We identify transcription factor ZNF263 as being significantly decreased in osimertinib-resistant or drug-tolerant persister LUAD cells and clinical residual tumors. ZNF263 overexpression improves the initial response of cells and delays the formation of persister cells with osimertinib treatment. We further show that ZNF263 binds and recruits DNMT1 to the EGFR gene promoter, suppressing EGFR transcription with DNA hypermethylation. ZNF263 interacts with nuclear EGFR, impairing the EGFR-STAT5 interaction to enhance AURKA expression. Overexpressing ZNF263 also makes tumor cells with wild-type EGFR expression or refractory EGFR mutations more susceptible to EGFR inhibition. More importantly, lentivirus or adeno-associated virus (AAV)-mediated ZNF263 overexpression synergistically suppresses tumor growth and regrowth with osimertinib treatment in xenograft animal models. These findings suggest that enhancing ZNF263 may achieve complete response in LUAD with EGFR-targeted therapies.

Retinoic acid receptor alpha inhibits ferroptosis by promoting thioredoxin and protein phosphatase 1F in lung adenocarcinoma.

Ferroptosis is a recently discovered form of cell death that plays an important role in tumor growth and holds promise as a target for antitumor therapy. However, evidence in the regulation of ferroptosis in lung adenocarcinoma (LUAD) remains elusive. Here, we show that retinoic acid receptor alpha (RARA) is upregulated with the treatment of ferroptosis inducers (FINs). Pharmacological activation of RARA increases the resistance of LUAD to ferroptosis according to cell viability and lipid peroxidation assays, while RARA inhibitor or knockdown (KD) does the opposite. Through transcriptome sequencing in RARA-KD cells and chromatin immunoprecipitation (CHIP)-Seq data, we identify thioredoxin (TXN) and protein phosphatase 1 F (PPM1F) as downstream targets of RARA, both of which inhibit ferroptosis. We confirm that RARA binds to the promotor region of TXN and PPM1F and promotes their transcription by CHIP-qPCR and dual-luciferase assays. Overexpression of TXN and PPM1F reverses the effects of RARA knockdown on ferroptosis in vitro and vivo. Clinically, RARA knockdown or inhibitor increases cells' sensitivity to pemetrexed and cisplatin (CDDP). Immunohistochemistry (IHC) of LUAD from our cohort shows the same expression tendency of RARA and the downstream targets. Our study uncovers that RARA inhibits ferroptosis in LUAD by promoting TXN and PPM1F, and inhibiting RARA-TXN/PPM1F axis represents a promising strategy for improving the efficacy of FINs or chemotherapy in the treatment of LUAD patients.

Targeting TAM-secreted S100A9 effectively enhances the tumor-suppressive effect of metformin in treating lung adenocarcinoma.

Metformin's effect on tumor treatment was complex, because it significantly reduced cancer cell proliferation in vitro, but made no difference in prognosis in several clinical cohorts. Our transcriptome sequencing results revealed that tumor-associated macrophage (TAM) infiltration significantly increased in active lung adenocarcinoma (LUAD) patients with long-term metformin use. We further identified that the tumor suppressive effect of metformin was more significant in mice after the depletion of macrophages, suggesting that TAMs might play an important role in metformin's effects in LUAD. Combining 10X Genomics single-cell sequencing of tumor samples, transcriptome sequencing of metformin-treated TAMs, and the ChIP-Seq data of the Encode database, we identified and validated that metformin significantly increased the expression and secretion of S100A9 of TAMs through AMPK-CEBP/ß pathway. For the downstream, S100A9 binds to RAGE receptors on the surface of LUAD cells, and then activates the NF-κB pathway to promote EMT and progression of LUAD, counteracting the inhibitory effect of metformin on LUAD cells. In cell-derived xenograft models (CDX) and patient-derived xenograft models (PDX) models, our results showed that neutralizing antibodies targeting TAM-secreted S100A9 effectively enhanced the tumor suppressive effect of metformin in treating LUAD. Our results will enable us to better comprehend the complex role of metformin in LUAD, and advance its clinical application in cancer treatment.

Polyamine-mediated ferroptosis amplification acts as a targetable vulnerability in cancer.

Targeting ferroptosis, an iron-dependent form of regulated cell death triggered by the lethal overload of lipid peroxides, in cancer therapy is impeded by our limited understanding of the intersection of tumour's metabolic feature and ferroptosis vulnerability. In the present study, arginine is identified as a ferroptotic promoter using a metabolites library. This effect is mainly achieved through arginine's conversion to polyamines, which exerts their potent ferroptosis-promoting property in an H2O2-dependent manner. Notably, the expression of ornithine decarboxylase 1 (ODC1), the critical enzyme catalysing polyamine synthesis, is significantly activated by the ferroptosis signal--iron overload--through WNT/MYC signalling, as well as the subsequent elevated polyamine synthesis, thus forming a ferroptosis-iron overload-WNT/MYC-ODC1-polyamine-H2O2 positive feedback loop that amplifies ferroptosis. Meanwhile, we notice that ferroptotic cells release enhanced polyamine-containing extracellular vesicles into the microenvironment, thereby further sensitizing neighbouring cells to ferroptosis and accelerating the "spread" of ferroptosis in the tumour region. Besides, polyamine supplementation also sensitizes cancer cells or xenograft tumours to radiotherapy or chemotherapy through inducing ferroptosis. Considering that cancer cells are often characterized by elevated intracellular polyamine pools, our results indicate that polyamine metabolism exposes a targetable vulnerability to ferroptosis and represents an exciting opportunity for therapeutic strategies for cancer.

The immunomodulatory role of all-trans retinoic acid in tumor microenvironment.

Retinoids are essential nutrients for human beings. Among them, all-trans retinoic acid (ATRA), considered one of the most active metabolites, plays important roles in multiple biological processes. ATRA regulates the transcription of target genes by interacting with nuclear receptors bonded to retinoic acid response elements (RAREs). Besides its differentiation-inducing effect in the treatment of acute promyelocytic leukemia and some solid tumor types, its immunoregulatory role in tumor microenvironment (TME) has attracted considerable attention. ATRA not only substantially abrogates the immunosuppressive effect of tumor-infiltrating myeloid-derived suppressor cells but also activates the anti-tumor effect of CD8 + T cells. Notably, the combination of ATRA with other therapeutic approaches, including immune checkpoint inhibitors (ICIs), tumor vaccines, and chemotherapy, has been extensively investigated in a variety of tumor models and clinical trials. In this review, we summarize the current understanding of the role of ATRA in cancer immunology and immunotherapy, dissect the underlying mechanisms of ATRA-mediated activation or differentiation of different types of immune cells, and explore the potential clinical significance of ATRA-based cancer therapy.

Hepatic nuclear factor 4 alpha promotes the ferroptosis of lung adenocarcinoma via transcriptional activation of cytochrome P450 oxidoreductase.

Background: Lung adenocarcinoma is one of the most prevalent cancers while ferroptosis is crucial for cancer therapies. This study aims to investigate the function and mechanism of hepatic nuclear factor 4 alpha (HNF4A) in lung adenocarcinomas' ferroptosis. Materials and Methods: HNF4A expression in ferroptotic A549 cells was detected. Then HNF4A was knocked down in A549 cells while overexpressed in H23 cells. Cells with changed HNF4A expression were tested for cytotoxicity and the level of cellular lipid peroxidation. The expression of cytochrome P450 oxidoreductase (POR) expression was examined after HNF4A was knocked down or overexpressed. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) and dual-luciferase assays were performed to validate the regulation of HNF4A on POR. Finally, POR was restored in HNF4A-altered cells to check whether it restores the effect of HNF4A on ferroptosis. Results: We found that HNF4A expression significantly decreased in the ferroptosis of A549 cells, and this change can be blocked by deferoxamine, an inhibitor of ferroptosis. Knockdown of HNF4A inhibited ferroptosis in A549 cells while overexpression of HNF4A promoted ferroptosis in H23 cells. We identified a key ferroptosis-related gene, POR serves as a potential target gene of HNF4A, whose expression was significantly changed in lung adenocarcinoma cells knocking down or overexpressing HNF4A. We demonstrated that HNF4A was bound to the POR's promoter to enhance POR expression, and identified the binding sites via ChIP-qPCR and luciferase assays. Restoration of POR expression blocked the promoting effect of HNF4A on ferroptosis in lung adenocarcinoma. Conclusion: HNF4A promotes POR expression through binding to the POR's promoter, and subsequently promotes the ferroptosis of lung adenocarcinoma.

PLK1 regulating chemoradiotherapy sensitivity of esophageal squamous cell carcinoma through pentose phosphate pathway/ferroptosis.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is the most common pathological type of esophageal cancer in China, accounting for more than 90 %. Most patients were diagnosed with advanced-stage ESCC, for whom new adjuvant therapy is recommended. Therefore, it is urgent to explore new therapeutic targets for ESCC. Ferroptosis, a newly discovered iron-dependent programmed cell death, has been shown to play an important role in carcinogenesis by many studies. This study explored the effect of Polo like kinase 1 (PLK1) on chemoradiotherapy sensitivity of ESCC through ferroptosis METHODS: In this study, we knocked out the expression of PLK1 (PLK1-KO) in ESCC cell lines (KYSE150 and ECA109) with CRISPR/CAS9. The effects of PLK1-knock out on G6PD, the rate-limiting enzyme of pentose phosphate pathway (PPP), and downstream NADPH and GSH were explored. The lipid peroxidation was observed by flow cytometry, and the changes in mitochondria were observed by transmission electron microscopy. Next, through the CCK-8 assay and clone formation assay, the sensitivity to cobalt 60 rays, paclitaxel, and cisplatin were assessed after PLK1-knock out, and the nude mouse tumorigenesis experiment further verified it. The regulation of transcription factor YY1 on PLK1 was evaluated by dual luciferase reporter assay. The expression and correlation of PLK1 and YY1, and their impact on prognosis were analyzed in more than 300 ESCC cases from the GEO database and our center. Finally, the above results were further proved by single-cell sequencing. RESULTS: After PLK1 knockout, the expression of G6PD dimer and the level of NADPH and GSH in KYSE150 and ECA109 cells significantly decreased. Accordingly, lipid peroxidation increased, mitochondria became smaller, membrane density increased, and ferroptosis was more likely to occur. However, with the stimulation of exogenous GSH (10 mM), there was no significant difference in lipid peroxidation and ferroptosis between the PLK1-KO group and the control group. After ionizing radiation, the PLK1-KO group had higher lipid peroxidation ratio, more cell death, and was more sensitive to radiation, while exogenous GSH (10 mM) could eliminate this difference. Similar results could also be observed when receiving paclitaxel combined with cisplatin and chemoradiotherapy. The expression of PLK1, G6PD dimer, and the level of NADPH and GSH in KYSE150, ECA109, and 293 T cells stably transfected with YY1-shRNAs significantly decreased, and the cells were more sensitive to radiotherapy and chemotherapy. ESCC patients from the GEO database and our center, YY1 and PLK1 expression were significantly positively-correlated, and the survival of patients with high expression of PLK1 was significantly shorter. Further analysis of single-cell sequencing specimens of ESCC in our center confirmed the above results. CONCLUSION: In ESCC, down-regulation of PLK1 can inhibit PPP, and reduce the level of NADPH and GSH, thereby promoting ferroptosis and improving their sensitivity to radiotherapy and chemotherapy. Transcription factor YY1 has a positive regulatory effect on PLK1, and their expressions were positively correlated. PLK1 may be a target for predicting and enhancing the chemoradiotherapy sensitivity of ESCC.

Characterization of Infiltrating Immune Cells and Secretory or Membrane-Associated Proteins in KRAS Lung Adenocarcinoma.

Background: This study identified the expression and prognosis significance of secretory or membrane-associated proteins in KRAS lung adenocarcinoma (LUAD) and depicted the characteristics between the immune cell infiltration and the expression of these genes. Methods: Gene expression data of LUAD samples (n = 563) were accessed from The Cancer Genome Atlas (TCGA). The expression of secretory or membrane-associated proteins was compared among the KRAS-mutant, wild-type, and normal groups, as well as the subgroup of the KRAS-mutant group. We identified the survival-related differentially expressed secretory or membrane-associated proteins and conducted the functional enrichment analysis. Then, the characterization and association between their expression and the 24 immune cell subsets were investigated. We also constructed a scoring model to predict KRAS mutation by LASSO and logistic regression analysis. Results: Secretory or membrane-associated genes with differential expression (n = 74) across three groups (137 KRAS LUAD, 368 wild-type LUAD, and 58 normal groups) were identified, and the results of GO and KEGG indicated that they were strongly associated with immune cell infiltrations. Among them, ten genes were significantly related to the survival of patients with KRAS LUAD. The expression of IL37, KIF2, INSR, and AQP3 had the most significant correlations with immune cell infiltration. In addition, eight DEGs from the KRAS subgroups were highly correlated with immune infiltrations, especially TNFSF13B. Using LASSO-logistic regression, a KRAS mutation prediction model based on the 74 differentially expressed secretory or membrane-associated genes was built, and the accuracy was 0.79. Conclusion: The research investigated the relationship between the expression of KRAS-related secretory or membrane-associated proteins in LUAD patients with prognostic prediction and immune infiltration characterization. Our study demonstrated that secretory or membrane-associated genes were closely associated with the survival of KRAS LUAD patients and were strongly correlated to immune cell infiltration.

HNF4G increases cisplatin resistance in lung adenocarcinoma via the MAPK6/Akt pathway.

Background: Lung adenocarcinoma is one of the most common tumors, and cisplatin is frequently used in treating lung adenocarcinoma patients. This study aimed to look into the roles and mechanisms of HNF4G in cisplatin resistance of lung adenocarcinoma. Materials & Methods: Cisplatin resistance and gene expression data of 542 cell lines from the CTRP and CCLE databases were analyzed. HNF4G expression was detected in the lung adenocarcinoma cell lines after treatment with various concentrations of cisplatin. Cisplatin sensitivity curves were detected in cells that overexpressed or knocked down HNF4G. The ChIP-Seq data were then analyzed to identify the targets of HNF4G involved in cisplatin resistance. Expression and phosphorylation of the MAPK6/Akt pathway were detected after HNF4G was overexpressed or knocked down. Finally, ChIP-qPCR and dual-luciferase assays were used to investigate the regulation of HNF4G on MAPK6. Results: In cell lines, high expression of HNF4G was significantly positively correlated with cisplatin resistance, and lung adenocarcinoma patients who had high HNF4G expression had a poor prognosis. Cisplatin treatment increased HNF4G expression, and overexpression of HNF4G significantly increased the resistance to cisplatin in A549 and HCC827 cells, whereas knockdown of HNF4G had the opposite effect. HNF4G overexpression increased MAPK6 expression and activated the MAPK6/Akt pathway, while an Akt inhibitor reduced the effects of HNF4G on cisplatin resistance. HNF4G bound to the MAPK6 promoter region, promoting MAPK6 expression, according to ChIP-qPCR and luciferase assays. Conclusion: By binding to the MAPK6 promoter region, HNF4G promotes MAPK6 expression and subsequent Akt phosphorylation, resulting in resistance to cisplatin in lung adenocarcinoma.

Identification of the relationship between single-cell N6-methyladenosine regulators and the infiltrating immune cells in esophageal carcinoma.

Background: N6-methyladenosine (m6A) RNA methylation plays a crucial role in important genomic processes in a variety of malignancies. However, the characterization of m6A with infiltrating immune cells in the tumor microenvironment (TME) in esophageal squamous carcinoma (ESCC) remains unknown. Methods: The single-cell transcriptome data from five ESCC patients in our hospital were analyzed, and TME clusters associated with prognosis and immune checkpoint genes were investigated. Cell isolation and qPCR were conducted to validate the gene characterization in different cells. Results: According to distinct biological processes and marker genes, macrophages, T cells, and B cells clustered into three to four different subgroups. In addition, we demonstrated that m6A RNA methylation regulators were strongly related to the clinical and biological features of ESCC. Analysis of transcriptome data revealed that m6A-mediated TME cell subsets had high predictive value and showed a close relationship with immune checkpoint genes. The validation results from qPCR demonstrated the characteristics of essential genes. CellChat analysis revealed that RNA from TME cells m6A methylation-associated cell subtypes had substantial and diversified interactions with cancer cells. Further investigation revealed that MIF- (CD74+CXCR4) and MIF- (CD74+CD44) ligand-receptor pairings facilitated communication between m6A-associated subtypes of TME cells and cancer cells. Conclusion: Overall, our study demonstrated for the first time the function of m6A methylation-mediated intercellular communication in the microenvironment of tumors in controlling tumor development and anti-tumor immune regulation in ESCC.