A lava-inspired proteolytic enzyme therapy on cancer with a PEG-based hydrogel enhances tumor distribution and penetration of liposomes.

The enhanced permeability and retention (EPR) effect has become the guiding principle for nanomedicine against cancer for a long time. However, several biological barriers severely resist therapeutic agents' penetration and retention into the deep tumor tissues, resulting in poor EPR effect and high tumor mortality. Inspired by lava, we proposed a proteolytic enzyme therapy to improve the tumor distribution and penetration of nanomedicine. A trypsin-crosslinked hydrogel (Trypsin@PSA Gel) was developed to maintain trypsin's activity. The hydrogel postponed trypsin's self-degradation and sustained the release. Trypsin promoted the cellular uptake of nanoformulations in breast cancer cells, enhanced the penetration through endothelial cells, and degraded total and membrane proteins. Proteomic analysis reveals that trypsin affected ECM components and down-regulated multiple pathways associated with cancer progression. Intratumoral injection of Trypsin@PSA Gel significantly increased the distribution of liposomes in tumors and reduced tumor vasculature. Combination treatment with intravenous injection of gambogic acid-loaded liposomes and intratumoral injection of Trypsin@PSA Gel inhibited tumor growth. The current study provides one of the first investigations into the enhanced tumor distribution of liposomes induced by a novel proteolytic enzyme therapy.

Oridonin, a small molecule inhibitor of cancer stem cell with potent cytotoxicity and differentiation potential.

Cancer stem cells (CSCs) drive malignant tumor progression, recurrence, and metastasis with unique characteristics, including self-renewal and resistance to conventional treatments. Conventional differentiation inducers, although promising, have limited cytotoxicity and may inadvertently enhance CSC stemness. To address these challenges, ongoing efforts are dedicated to developing strategies that can effectively combine both cytotoxicity and differentiation-inducing effects. In this study, we introduce oridonin (Ori), a small molecule with dual differentiation-inducing and cytotoxicity properties capable of eliminating tumor CSCs. We isolated CSCs in B16F10 cells using the Hoechst side population method and assessed the differentiation effect of Ori. Ori's differentiation-inducing effect was further evaluated using human acute promyelocytic leukemia. The cytotoxic potential of Ori against MCF-7 and B16F10 cell lines was assessed through various methods. In vivo anti-tumor and anti-CSC efficacy of Ori was investigated using mouse melanoma and CSCs melanoma models. Safety evaluation included zebrafish embryotoxicity and mouse acute toxicity experiments. As a result, Ori effectively dismantles tumorspheres, inhibits proliferation, and reduces the expression of CSC-specific markers. It induces significant differentiation, especially in the case of NB4. Additionally, Ori upregulates TP53 expression, mitigates the hypoxic tumor microenvironment, suppresses stemness, and inhibits PD-L1 expression, prompting a robust anti-cancer immune response. Ori demonstrates pronounced cytotoxicity, inducing notable pro-apoptotic effects on B16F10 and MCF-7 cells, with specific triggering of mitochondrial apoptosis. Importantly, Ori maintains a commendable biosafety record. The dual-action prowess of Ori not only induces the differentiation of CSCs but also dispatches differentiated and residual tumor cells, effectively thwarting the relentless march of tumor progression.

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.

IL6-STAT3-C/EBPß-IL6 positive feedback loop in tumor-associated macrophages promotes the EMT and metastasis of lung adenocarcinoma.

BACKGROUND: Lung cancer is one of the most common tumors in the world, and metastasis is one of the major causes of tumor-related death in lung cancer patients. Tumor-associated macrophages (TAMs) are a major component of the tumor microenvironment (TME) and are frequently associated with tumor metastasis in human cancers. However, the regulatory mechanisms of TAMs in lung cancer metastasis remain unclear. METHODS: Single-cell sequencing analysis of lung cancer and normal tissues from public databases and from 14 patients who underwent surgery at Zhongshan Hospital was performed. In vitro co-culture experiments were performed to evaluate the effects of TAMs on lung cancer migration and invasion. Changes in the expression of IL-6, STAT3, C/EBPΒ, and EMT pathway were verified using RT-qPCR, western blotting, and immunofluorescence. Dual luciferase reporter assays and ChIP were used to reveal potential regulatory sites on the transcription factor sets. In addition, the effects of TAMs on lung cancer progression and metastasis were confirmed by in vivo models. RESULTS: TAM infiltration is associated with tumor progression and poor prognosis. IL-6 secreted by TAMs can activate the JAK2/STAT3 pathway through autocrine secretion, and STAT3 acts as a transcription factor to activate the expression of C/EBPß, which further promotes the transcription and expression of IL-6, forming positive feedback loops for IL6-STAT3-C/EBPß-IL6 in TAMs. IL-6 secreted by TAMs promotes lung cancer progression and metastasis in vivo and in vitro by activating the EMT pathway, which can be attenuated by the use of JAK2/STAT3 pathway inhibitors or IL-6 monoclonal antibodies. CONCLUSIONS: Our data suggest that TAMs promote IL-6 expression by forming an IL6-STAT3-C/EBPß-IL6 positive feedback loop. Released IL-6 can induce the EMT pathway in lung cancer to enhance migration, invasion, and metastasis. The use of IL-6-neutralizing antibody can partially counteract the promotion of LUAD by TAMs. A novel mechanism of macrophage-promoted tumor progression was revealed, and the IL6-STAT3-C/EBPß-IL6 signaling cascade may be a potential therapeutic target against lung cancer.

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.

Global prevalence of polypharmacy and potentially inappropriate medication in older patients with dementia: a systematic review and meta-analysis.

Background: Older patients with dementia always need multiple drugs due to comorbidities and cognitive impairment, further complicating drug treatment and increasing the risk of potentially inappropriate medication. The objective of our study is to estimate the global prevalence of polypharmacy and potentially inappropriate medication (PIM) and explore the factors of PIM for older patients with dementia. Methods: We searched PubMed, Embase (Ovid), and Web of Science databases to identify eligible studies from inception to 16 June 2023. We conducted a meta-analysis for observational studies reporting the prevalence of potentially inappropriate medication and polypharmacy in older patients with dementia using a random-effect model. The factors associated with PIM were meta-analyzed. Results: Overall, 62 eligible studies were included, of which 53 studies reported the prevalence of PIM and 28 studies reported the prevalence of polypharmacy. The pooled estimate of PIM and polypharmacy was 43% (95% CI 38-48) and 62% (95% CI 52-71), respectively. Sixteen studies referred to factors associated with PIM use, and 15 factors were further pooled. Polypharmacy (2.83, 95% CI 1.80-4.44), diabetes (1.31, 95% CI 1.04-1.65), heart failure (1.17, 95% CI 1.00-1.37), depression (1.45, 95% CI 1.14-1.88), history of cancer (1.20, 95% CI 1.09-1.32), hypertension (1.46, 95% CI 1.05-2.03), ischemic heart disease (1.55, 95% CI 0.77-3.12), any cardiovascular disease (1.11, 95% CI 1.06-1.17), vascular dementia (1.09, 95% CI 1.03-1.16), chronic obstructive pulmonary disease (1.39, 95% CI 1.13-1.72), and psychosis (1.91, 95% CI 1.04-3.53) are positively associated with PIM use. Conclusion: PIM and polypharmacy were highly prevalent in older patients with dementia. Among different regions, the pooled estimate of PIM use and polypharmacy varied widely. Increasing PIM in older patients with dementia was closely associated with polypharmacy. For other comorbidities such as heart failure and diabetes, prescribing should be cautioned.

AGRN promotes lung adenocarcinoma progression by activating Notch signaling pathway and acts as a therapeutic target.

Lung cancer is the main reason for cancer-associated death globally, and lung adenocarcinoma (LUAD) is the most prevalent subtype of lung cancer. Recently, AGRN is considered playing an vital role in the development of some cancers. However, the regulatory effects and mechanisms of AGRN in LUAD remain elusive. In this study, we clarified the significant upregulation of AGRN expression in LUAD by single-cell RNA sequencing combined with immunohistochemistry. Besides, we confirmed that LUAD patients with high AGRN expression are more susceptible to lymph node metastases and have a worse prognosis by a retrospective study of 120 LUAD patients. Next, we demonstrated that AGRN directly interact with NOTCH1, which results in the release of the intracellular structural domain of NOTCH1 and the subsequent activation of the NOTCH pathway. Moreover, we also found that AGRN promotes proliferation, migration, invasion, EMT and tumorigenesis of LUAD cells in vitro and in vivo, and that these effects are reversed by blocking the NOTCH pathway. Furthermore, we prepared several antibodies targeting AGRN, and clarify that Anti-AGRN antibody treatment could significantly inhibit proliferation and promote apoptosis of tumor cells. Our study highlights the important role and regulatory mechanism of AGRN in LUAD development and progression, and suggests that antibodies targeting AGRN have therapeutic potential for LUAD. We also provide theoretical and experimental evidence for further development of monoclonal antibodies targeting AGRN.

Retinol Saturase Mediates Retinoid Metabolism to Impair a Ferroptosis Defense System in Cancer Cells.

Ferroptosis is an iron-dependent form of regulated cell death induced by the lethal overload of lipid peroxides in cellular membranes. In recent years, modulating ferroptosis has gained attention as a potential therapeutic approach for tumor suppression. In the current study, retinol saturase (RETSAT) was identified as a significant ferroptosis mediator using a publicly accessible CRISPR/Cas9 screening dataset. RETSAT depletion protected tumor cells from lipid peroxidation and subsequent cell death triggered by various ferroptosis inducers. Furthermore, exogenous supplementation with retinoids, including retinol (the substrate of RETSAT) and its derivatives retinal and retinoic acid, also suppressed ferroptosis, whereas the product of RETSAT, 13, 14-dihydroretinol, failed to do so. As effective radical-trapping antioxidant, retinoids protected the lipid membrane from autoxidation and subsequent fragmentation, thus terminating the cascade of ferroptosis. Pseudotargeted lipidomic analysis identified an association between retinoid regulation of ferroptosis and lipid metabolism. Retinoic acid, but not 13, 14-dihydroretinoic acid, interacted with its nuclear receptor and activated transcription of stearoyl-CoA desaturase, which introduces the first double bond into saturated fatty acid and thus catalyzes the generation of monounsaturated fatty acid, a known ferroptosis suppressor. Therefore, RETSAT promotes ferroptosis by transforming retinol to 13, 14-dihydroretinol, thereby turning a strong anti-ferroptosis regulator into a relatively weak one. SIGNIFICANCE: Retinoids have ferroptosis-protective properties and can be metabolized by RETSAT to promote ferroptosis, suggesting the possibility of targeting retinoid metabolism in cancer as a treatment strategy to trigger ferroptosis.

Dissecting the single-cell transcriptome network of macrophage and identifies a signature to predict prognosis in lung adenocarcinoma.

PURPOSE: The tumor immune microenvironment (TME) plays a vital role in tumorigenesis, progression, and treatment. Macrophages, as an important component of the tumor microenvironment, play an essential role in antitumor immunity and TME remodeling. In this study, we aimed to explore the different functions of different origins macrophages in TME and their value as potential predictive markers of prognosis and treatment. METHODS: We performed single-cell analysis using 21 lung adenocarcinoma (LUAD), 12 normal, and four peripheral blood samples from our data and public databases. A prognostic prediction model was then constructed using 502 TCGA patients and explored the potential factors affecting prognosis. The model was validated using data from 4 different GEO datasets with 544 patients after integration. RESULTS: According to the source of macrophages, we classified macrophages into alveolar macrophages (AMs) and interstitial macrophages (IMs). AMs mainly infiltrated in normal lung tissue and expressed proliferative, antigen-presenting, scavenger receptors genes, while IMs occupied the majority in TME and expressed anti-inflammatory, lipid metabolism-related genes. Trajectory analysis revealed that AMs rely on self-renew, whereas IMs originated from monocytes in the blood. Cell-to-cell communication showed that AMs interacted mainly with T cells through the MHC I/II signaling pathway, while IMs mostly interacted with tumor-associated fibrocytes and tumor cells. We then constructed a risk model based on macrophage infiltration and showed an excellent predictive power. We further revealed the possible reasons for its potential prognosis prediction by differential genes, immune cell infiltration, and mutational differences. CONCLUSION: In conclusion, we investigated the composition, expression differences, and phenotypic changes of macrophages from different origins in lung adenocarcinoma. In addition, we developed a prognostic prediction model based on different macrophage subtype infiltration, which can be used as a valid prognostic biomarker. New insights were provided into the role of macrophages in the prognosis and potential treatment of LUAD patients.

Postsurgical wound management and prevention of triple-negative breast cancer recurrence with a pryoptosis-inducing, photopolymerizable hydrogel.

Surgical removal remains the predominant treatment strategy for triple-negative breast cancer (TNBC). However, risks that include high locoregional recurrence and remote metastasis threaten patient survival and quality of life after surgery. In this study, a hydrogel based on poly (ethylene glycol) dimethacrylate and sericin methacryloyl was fabricated by photopolymerization to fill the resection cavity and prevent recurrence. The obtained hydrogel exhibited mechanical properties compatible with breast tissue and facilitated postsurgical wound management by promoting tissue regeneration. The DNA methylation inhibitor decitabine (DEC) and poly (lactic-co-glycolic acid)-encapsulated phytochemical gambogic acid (GA) were loaded into the hydrogel. The as-prepared hydrogel promoted fast release of DEC and sustained release of GA, leading to gasdermin E-mediated tumor cell pyroptosis and activating antitumor immune responses. Inducing postsurgical tumor cell pyroptosis inhibited local tumor recurrence and lung metastasis. While the dual-drug-loaded hydrogel system cured less than half of tumor-bearing mice, the cured mice survived for over half a year. These findings indicate that our hydrogel system is an excellent biocompatible platform for postsurgical TNBC therapy.

Cardiotoxicity with human epidermal growth factor receptor-2 inhibitors in breast cancer: Disproportionality analysis of the FDA adverse event reporting system.

BACKGROUND: The cardiotoxicity induced by human epidermal growth factor receptor-2 (HER-2) inhibitors in patients with breast cancer has been reported widely. However, these data sources were largely limited to fewer patients in clinical trials and case reports, lacking more comprehensive analysis from real-world data. METHODS: The cases diagnosed with breast cancer from January 2004 to December 2021 were extracted from the FDA adverse event database and further divided into 3 groups (the HER-2 inhibitor group, the positive control group, and the control group). The association between HER-2 inhibitors and cardiovascular adverse events was evaluated using the reporting odds ratio (ROR), a disproportionality method. RESULTS: A total of 167,639 breast cancer patients were included, including 18,615 cases in the HER-2 inhibitor drug group, 2568 cases in the positive control group, and 146,456 cases in the control group. A total of 2529 cases (13.5%) treated with HER-2 inhibitors experienced cardiovascular adverse events, mainly reported by health professionals (81.5%). The disproportionality analysis showed that cardiomyopathy was observed in all HER-2 inhibitors except trastuzumab deruxtecan. Trastuzumab-related CVAEs were most frequently reported (N =2075), and the median time was 80.50 days (IQR: 8.00 to 206.75 days). CONCLUSION: Based on real-world data analysis, our study demonstrated a significant association between HER-2 inhibitors and cardiovascular toxicity. Cardiac function in patients with breast cancer should be monitored early during anti-HER therapy, especially within six months.

Association between glycosylated hemoglobin levels, diabetes duration, and left ventricular diastolic dysfunction in patients with type 2 diabetes and preserved ejection fraction: a cross-sectional study.

Background: We aimed to explore the intricate interplay between glycated hemoglobin (HbA1C) levels, disease duration, and left ventricular diastolic dysfunction in patients with type 2 diabetes mellitus (T2DM) characterized by preserved ejection fraction. Methods: A cross-sectional study was conducted at the Second Affiliated Hospital of Hebei Medical University from January 2022 to December 2022. A total of 114 inpatients from the Department of Endocrinology were randomly selected based on the inclusion and exclusion criteria. Patients with T2DM were stratified into three subgroups, each comprising 38 patients, based on disease duration and HbA1C levels. A sub-analysis was conducted to explore variations among these three distinct groups. A control group comprised 38 age, gender, body mass index (BMI), and smoking habit-matched healthy volunteers form the Physical Examination Center of the same hospital. General demographic information, biochemical results, and echocardiographic data were collected, and correlation and linear regression analyses were performed. Results: Diabetic patients exhibited lower E/A values (0.85 (0.72, 1.17) vs. 1.20 (0.97, 1.30)) and elevated E/e' values (9.50 (8.75, 11.00) vs. 9.00 (7.67, 9.85)) compared to their normal controls. In the subgroup analysis, patients with a disease duration exceeding 2 years displayed reduced E/A values (0.85 (0.75, 1.10) vs. 1.10 (0.80, 1.30)) and elevated E/e' values (9.80 (9.20, 10.80) vs. 8.95 (7.77, 9.50)) in comparison to those with a disease duration of ≤2 years, p<0.05. Among patients with a disease duration surpassing 2 years, those with higher HbA1C levels exhibited lower E/A values (0.80 (0.70, 0.90) vs. (0.85 (0.75, 1.10)) and higher E/e' values (11.00 (9.87, 12.15) vs. 9.80 (9.20, 10.80)) in contrast to patients with low HbA1C levels, p<0.05. Multiple linear regression analysis identified HbA1C (ß=0.294, p<0.001) and disease duration (ß=0.319, p<0.001) as independent risk factors for the E/A value in diabetes patients. Furthermore, HbA1C (ß=0.178, p=0.015) and disease duration (ß=0.529, p<0.001) emerged as independent risk factors for the E/e' value in diabetic patients. Conclusions: In individuals with T2DM exhibiting preserved ejection fraction, the presence of left ventricular diastolic dysfunction is significantly associated with HbA1C levels and the duration of diabetes.

Atorvastatin ameliorated myocardial fibrosis in db/db mice by inhibiting oxidative stress and modulating macrophage polarization.

BACKGROUND: People with diabetes mellitus (DM) suffer from multiple chronic complications due to sustained hyperglycemia, especially diabetic cardiomyopathy (DCM). Oxidative stress and inflammatory cells play crucial roles in the occurrence and progression of myocardial remodeling. Macrophages polarize to two distinct phenotypes: M1 and M2, and such plasticity in phenotypes provide macrophages various biological functions. AIM: To investigate the effect of atorvastatin on cardiac function of DCM in db/db mice and its underlying mechanisms. METHODS: DCM mouse models were established and randomly divided into DM, atorvastatin, and metformin groups. C57BL/6 mice were used as the control. Cardiac function was evaluated by echocardiography. Hematoxylin and eosin and Masson staining was used to examine the morphology and collagen fibers in myocardial tissues. The expression of transforming growth factor-ß1 (TGF-ß1), tumor necrosis factor-α (TNF-α), interleukin-1 ß (IL-1ß),M1 macrophages (iNOS+), and M2 macrophages (CD206+) were demonstrated by immunohistochemistry and immunofluorescence staining. The levels of TGF-ß1, IL-1ß, and TNF-α were detected by ELISA and real-time quantitative polymerase chain reaction. Malondialdehyde (MDA) concentrations and superoxide dismutase (SOD) ac-tivities were also measured. RESULTS: Treatment with atorvastatin alleviated cardiac dysfunction and decreased db/db mice. The broken myocardial fibers and deposition of collagen in the myocardial interstitium were relieved especially by atorvastatin treatment. Atorvastatin also reduced the levels of serum lactate dehydrogenase, creatine kinase isoenzyme, and troponin; lowered the levels of TGF-ß1, TNF-α and IL-1ß in serum and myocardium; decreased the concentration of MDA and increased SOD activity in myocardium of db/db mice; inhibited M1 macrophages; and promoted M2 macrophages. CONCLUSION: Administration of atorvastatin attenuates myocardial fibrosis in db/db mice, which may be associated with the antioxidative stress and anti-inflammatory effects of atorvastatin on diabetic myocardium through modulating macrophage polarization.

Upregulation of KLHL17 promotes the proliferation and migration of non-small cell lung cancer by activating the Ras/MAPK signaling pathway.

Analysis of the Gene Expression Profiling Interactive Analysis (GEPIA) database revealed that Kelch-like 17 (KLHL17) is overexpressed in non-small cell lung cancer (NSCLC) including adenocarcinoma (ADC) and squamous cell carcinoma (SCC). We therefore explored the role of KLHL17 in the development and progression of NSCLC. Immunohistochemistry and western blotting showed that KLHL17 expression was significantly higher in the tumor tissues from 173 patients with NSCLC, compared with the corresponding non-neoplastic tissue. In addition, upregulated KLHL17 expression was positively correlated with tumor size, lymph node metastasis and tumor node metastasis (TNM) stage, and affected the overall survival (OS) of patients with NSCLC. Consistent with clinical samples, in vitro studies demonstrated that KLHL17 expression was higher in various cell lines of NSCLC (A549, H1299, H460 and SK cells) as compared to normal human bronchial epithelial cells (HBE cells). Overexpression of KLHL17 in the cell lines of NSCLC with KLHL17-Flag plasmid promoted the proliferation and migration of tumor cells, which was associated with elevated activation of Rat sarcoma/Mitogen-activated protein kinases (Ras/MAPK) signaling and increased expression of cyclin D1, cyclin D-dependent kinases 4 (CDK4), matrix metalloproteinase 2 (MMP2) and Ras homolog gene family member A (RhoA). In contrast, knockdown of KLHL17 in the cell lines of NSCLC using KLHL17 small interfering RNA suppressed the proliferation and migration of tumor cells, in association with reduced activation of Ras/MAPK signaling and decreased expression of cyclin D1, CDK4, MMP2 and RhoA. Moreover, treatment of tumor cells with Ras inhibitor salirasib prevented KLHL17-induced Ras/MAPK activity as well as tumor proliferation and migration. These results suggest that upregulated KLHL17 in NSCLC promotes the proliferation and migration of tumor by activating Ras/MAPK signaling pathway. Therefore, KLHL17 may be a novel therapeutic target for the treatment of NSCLC.

Increasing Trends of Polypharmacy and Potentially Inappropriate Medication Use in Older Lung Cancer Patients in China: A Repeated Cross-Sectional Study.

Objectives: Polypharmacy and potentially inappropriate medication (PIM) use are frequent in older lung cancer patients. This study aimed to examine the trends of polypharmacy and PIM use and explore risk factors for PIM use based on the 2019 Beers criteria in older Chinese lung cancer outpatients with multimorbidity. Methods: A repeated cross-sectional study was conducted using electronic medical data consisting of the prescriptions of older lung cancer outpatients in China from January 2016 to December 2018. Polypharmacy was defined as the use of five or more medications. The 2019 Beers criteria were used to evaluate the PIM use of older cancer outpatients (age ≥65 years), and multivariate logistic regression was used to identify the risk factors for PIM use. Results: A total of 3,286 older lung cancer outpatients and their prescriptions were included in the study. The prevalence of polypharmacy was 14.27% in 2016, 16.55% in 2017, and 18.04% in 2018. The prevalence of PIM use, according to the 2019 Beers criteria, was 31.94% in 2016, 35.78% in 2017, and 42.67% in 2018. The two most frequently used PIMs in older lung cancer outpatients were estazolam and tramadol. The logistic regression demonstrated that age 75 to 79, polypharmacy, irrational use of drugs, and lung cancer accompanied by sleep disorders, anxiety or depression, or pain were positively associated with PIM use in older lung cancer outpatients. Conclusion: The prevalence of polypharmacy and PIM use in older lung cancer outpatients with multimorbidity was high in China, and polypharmacy and PIM use increased over time. Further research on interventions rationing PIM use in the older lung cancer patient population is needed.

A Novel Prognostic Signature Revealed the Interaction of Immune Cells in Tumor Microenvironment Based on Single-Cell RNA Sequencing for Lung Adenocarcinoma.

Background: The tumor immune microenvironment (TIME) played an important role in immunotherapy prognosis and treatment response. Immune cells constitute a large part of the tumor microenvironment and regulate tumor progression. Our research is dedicated to studying the infiltrating immune cell in lung adenocarcinoma (LUAD) and seeking potential targets. Methods: The scRNA-seq data were collected from our FDZSH and two public datasets. The code for cell-type mapping algorithms was downloaded from the CIBERSORTx portal. The bioinformatics data of LUAD patients could be approached from The Cancer Genome Atlas (TCGA) portal. Weighted gene coexpression network analysis (WGCNA) and least absolute shrinkage and selection operator (LASSO) analyses were performed to construct a risk model. TIMER2 and TIDE helped with the immune infiltration estimation, while PROGENy helped the cancer-related pathways' enrichment analysis. GSE31210 dataset and IMVigor ICB therapy cohort validated our findings as the external validation datasets. Results: We clustered the scRNA-seq dataset (integrating our FDZSH datasets and other public datasets) into 23 subpopulations. After curated cell annotation, we implemented Cibersort and WGCNA analysis to anchor the brown module and natural killer cell cluster1 due to the most relationship with tumor trait. The overlap of the brown module gene, natural killer cell signature, and DEGs of tumor and adjacent normal samples was screened by LASSO Cox regression. The obtained 5-gene risk model showed an excellent prognostic performance in the validation dataset. Furthermore, there was a correlation between risk score and tumor-infiltrating immune cells and tumor genomics abnormity. Patients with higher risk scores had a significantly lower immunotherapy response rate. Conclusion: Our observations implied that immune cells played a pivotal role in TIME and established a 5-gene signature (including IDH2, ADRB2, SFTPC, CCDC69, and CCND2) on the basement of nature killer markers targeted by WGCNA analysis. The significance of clinical outcome and immunotherapy response prediction was validated robustly.

Weighted Correlation Network Analysis of Cancer Stem Cell-Related Prognostic Biomarkers in Esophageal Squamous Cell Carcinoma.

Background: The role of cancer stem cells in esophageal squamous cell carcinoma (ESCC) remains unclear. Methods: The mRNA stemness index (mRNAsi) of 179 ESCC patients (GSE53625) was calculated using a machine learning algorithm based on their mRNA expression. Stemness-related genes were identified by weighted correlation network analysis (WGCNA) and LASSO regression, whose associations with mutation status, immune cell infiltrations, and potential compounds were also analyzed. The role of these genes in proliferation and their expressions was assessed in ESCC cell lines and 112 samples from our center. Results: The ESCC samples had significantly higher mRNAsi than the normal tissues. Patients with high mRNAsi exhibited higher worse OS. Seven stemness-related genes were identified by WGCNA and LASSO regression, based on which a risk-predicted score model was constructed. Among them, CST1, CILP, PITX2, F2RL2, and RIOX1 were favorable for OS, which were adverse for DPP4 and ZFHX4 in the GSE53625 dataset. However, RIOX1 was unfavorable for OS in patients from our center. In vitro assays showed that CST1, CILP, PITX2, F2RL2, and RIOX1 were pro-proliferated, which were opposite for DDP4 and ZFHX4. In addition, SMARCA4, NOTCH3, DNAH5, and KALRN were more mutated in the low-score group. The low-score group had significantly more memory B cells, monocytes, activated NK cells, and Tregs and less macrophages M2, resting mast cells, and resting dendritic cells. Conclusions: Seven stemness-related genes are significantly related to the prognosis, gene mutations, and immune cell infiltration of ESCC. Some potential anticancer compounds may be favorable for OS.

Prescription of Potentially Inappropriate Medication Use in Older Cancer Outpatients With Multimorbidity: Concordance Among the Chinese, AGS/Beers, and STOPP Criteria.

Objectives: Age-related multimorbidity is a general problem in older patients, which increases the prevalence of potentially inappropriate medication (PIM) use. This study aimed to examine the prevalence and predictors of PIM use in older Chinese cancer outpatients with multimorbidity based on the 2017 Chinese criteria, 2019 AGS/Beers criteria, and 2014 STOPP criteria. Methods: A cross-sectional study was conducted using electronic medical data from nine tertiary hospitals in Chengdu from January 2018 to December 2018. The 2017 Chinese criteria, 2019 AGS/Beers criteria, and 2014 STOPP criteria were used to evaluate the PIM status of older cancer outpatients (age ≥65 years), the concordance among the three PIM criteria was calculated using kappa tests, and multivariate logistic regression was used to identify the risk factors associated with PIM use. Results: A total of 6,160 cancer outpatient prescriptions were included in the study. The prevalence of PIM use was 34.37, 32.65, and 15.96%, according to the 2017 Chinese criteria, 2019 AGS/Beers criteria, and 2014 STOPP criteria, respectively. Furthermore, 62.43% of PIMs met table 2, 0.27% of PIMs met table 3, 34.68% of PIMs met table 4, 2.62% of PIMs met table 5 of 2019 AGS/Beers criteria, respectively. According to the three criteria, 84.93%, 82.25%, and 94.61% of older cancer outpatients had one PIM. The most frequently used PIM in cancer outpatients was estazolam. The Chinese criteria and the STOPP criteria indicated poor concordance, whereas the 2019 AGS/Beers criteria showed moderate concordance with the other two criteria. Logistic regression demonstrated that age ≥ 80, more diseases, polypharmacy, irrational use of drugs, and lung cancer were positively associated with PIM use in older cancer outpatients. Conclusion: The prevalence of PIM use in Chinese older cancer outpatients with multimorbidity is high in China, and poor-to-moderate concordance among the three criteria was observed. Research on building PIM criteria for the older cancer population is necessary in the future.

miR-6077 promotes cisplatin/pemetrexed resistance in lung adenocarcinoma via CDKN1A/cell cycle arrest and KEAP1/ferroptosis pathways.

Lung adenocarcinoma (LUAD) is one of the most common malignancies worldwide. Combination chemotherapy with cisplatin (CDDP) plus pemetrexed (PEM) remains the predominant therapeutic regimen; however, chemoresistance greatly limits its curative potential. Here, through CRISPR-Cas9 screening, we identified miR-6077 as a key driver of CDDP/PEM resistance in LUAD. Functional experiments verified that ectopic overexpression of miR-6077 desensitized LUAD cells to CDDP/PEM in both cell lines and patient-derived xenograft models. Through RNA sequencing in cells and single-cell sequencing of samples from patients with CDDP/PEM treatments, we observed CDDP/PEM-induced upregulation of CDKN1A and KEAP1, which in turn activated cell-cycle arrest and ferroptosis, respectively, thus leading to cell death. Through miRNA pull-down, we identified and validated that miR-6077 targets CDKN1A and KEAP1. Furthermore, we demonstrated that miR-6077 protects LUAD cells from cell death induced by CDDP/PEM via CDKN1A-CDK1-mediated cell-cycle arrest and KEAP1-NRF2-SLC7A11/NQO1-mediated ferroptosis, thus resulting in chemoresistance in multiple LUAD cells both in vitro and in vivo. Moreover, we found that GMDS-AS1 and LINC01128 sensitized LUAD cells to CDDP/PEM by sponging miR-6077. Collectively, these results imply the critical role of miR-6077 in LUAD's sensitivity to CDDP/PEM, thus providing a novel therapeutic strategy for overcoming chemoresistance in clinical practice.

Sirtuin 2 Alleviates Chronic Neuropathic Pain by Suppressing Ferroptosis in Rats.

Neuropathic pain (NP) is chronic and associated with poor effects of general analgesia. It affects patients' health and quality of life. The apoptotic process of lipid peroxidation caused by iron overload is called ferroptosis, which may be associated with nervous system disease. A recent study has found that sirtuin 2 (SIRT2) achieves a neuroprotective effect by suppressing ferroptosis. Herein, we aimed to examine whether SIRT2 regulated spared nerve injury (SNI)-induced NP by suppressing ferroptosis in rats. A rat model of NP was induced in adult male Sprague-Dawley rats weighing 200-250 g. Mechanical allodynia was observed from the first day after SNI and continued for 14 days. Compared with age-matched control rats, the expression of SIRT2 and ferroportin 1 (FPN1) decreased in the L4-6 spinal cord of the SNI-induced NP rats. In addition, we observed that the levels of both iron and anti-acyl-coenzyme A synthetase long-chain family member 4 (ACSL4) were significantly increased in the spinal cord after SNI, while the expression of glutathione peroxidase 4 (GPX4) was decreased. Furthermore, an intrathecal injection of SIRT2 overexpressed recombinant adenovirus, which upregulated the expression of SIRT2, attenuated mechanical allodynia, enhanced the level of FPN1, inhibited intracellular iron accumulation, and reduced oxidant stress levels, thereby reversing the changes to ACSL4 and GPX4 expression in the SNI rats. This evidence suggests that SIRT2-targeted therapeutics may help relieve the symptoms of chronic NP.