The deubiquitinase USP2a promotes tumor immunosuppression by stabilizing immune checkpoint B7-H4 in lung adenocarcinoma harboring EGFR-activating mutants.

B7-H4 is an immune checkpoint crucial for inhibiting CD8+ T-cell activity. A clinical trial is underway to investigate B7-H4 as a potential immunotherapeutic agent. However, the regulatory mechanism of B7-H4 degradation via the ubiquitin-proteasome pathway (UPP) remains poorly understood. In this study, we discovered that proteasome inhibitors effectively increased B7-H4 expression, while EGFR-activating mutants promoted B7-H4 expression through the UPP. We screened B7-H4 binding proteins by co-immunoprecipitation and mass spectrometry and found that USP2a acted as a deubiquitinase of B7-H4 by removing K48- and K63-linked ubiquitin chains from B7-H4, leading to a reduction in B7-H4 degradation. EGFR mutants enhanced B7-H4 stability by upregulating USP2a expression. We further investigated the role of USP2a in tumor growth in vivo. Depletion of USP2a in L858R/LLC cells inhibited tumor cell proliferation, consequently suppressing tumor growth in immune-deficient nude mice by destabilizing downstream molecules such as Cyclin D1. In an immune-competent C57BL/6 mouse tumor model, USP2a abrogation facilitated infiltration of CD95+CD8+ effector T cells and hindered infiltration of Tim-3+CD8+ and LAG-3+CD8+ exhausted T cells by destabilizing B7-H4. Clinical lung adenocarcinoma samples showed a significant correlation between B7-H4 abundance and USP2a expression, indicating the contribution of the EGFR/USP2a/B7-H4 axis to tumor immunosuppression. In summary, this study elucidates the dual effects of USP2a in tumor growth by stabilizing Cyclin D1, promoting tumor cell proliferation, and stabilizing B7-H4, contributing to tumor immunosuppression. Therefore, USP2a represents a potential target for tumor therapy.

Opioid-free anaesthesia reduces postoperative nausea and vomiting after thoracoscopic lung resection: a randomised controlled trial.

BACKGROUND: Intraoperative opioid use has a positive relationship with postoperative nausea and vomiting (PONV), and opioid-free anaesthesia (OFA) might reduce PONV. We investigated whether OFA compared with opioid-based anaesthesia would reduce PONV during the first 2 postoperative days among patients undergoing thoracoscopic lung resection. METHODS: In this randomised controlled trial, 120 adult patients were randomly assigned (1:1, stratified by sex) to receive either OFA with esketamine, dexmedetomidine, and sevoflurane, or opioid-based anaesthesia with sufentanil and sevoflurane. A surgical pleth index (SPI) of 20-50 was applied for intraoperative analgesia provision. All subjects received PONV prophylaxis (dexamethasone and ondansetron) and multimodal analgesia (flurbiprofen axetil, ropivacaine wound infiltration, and patient-controlled sufentanil). The primary outcome was the occurrence of PONV during the first 48 h after surgery. RESULTS: The median age was 53 yr and 66.7% were female. Compared with opioid-based anaesthesia, OFA significantly reduced the incidence of PONV (15% vs 31.7%; odds ratio [OR]=0.38, 95% confidence interval [CI], 0.16-0.91; number needed to treat, 6; P=0.031). Secondary and safety outcomes were comparable between groups, except that OFA led to a lower rate of vomiting (OR=0.23, 95% CI, 0.08-0.77) and a longer length of PACU stay (median difference=15.5 min, 95% CI, 10-20 min). The effects of OFA on PONV did not differ in the prespecified subgroups of sex, smoking status, and PONV risk scores. CONCLUSIONS: In the context of PONV prophylaxis and multimodal analgesia, SPI-guided opioid-free anaesthesia halved the incidence of PONV after thoracoscopic lung resection, although it was associated with a longer stay in the PACU. CLINICAL TRIAL REGISTRATION: Chinese Clinical Trial Registry (ChiCTR2200059710).

Multi-omics analyses reveal spatial heterogeneity in primary and metastatic oesophageal squamous cell carcinoma.

BACKGROUND: Biopsies obtained from primary oesophageal squamous cell carcinoma (ESCC) guide diagnosis and treatment. However, spatial intra-tumoral heterogeneity (ITH) influences biopsy-derived information and patient responsiveness to therapy. Here, we aimed to elucidate the spatial ITH of ESCC and matched lymph node metastasis (LNmet ). METHODS: Primary tumour superficial (PTsup ), deep (PTdeep ) and LNmet subregions of patients with locally advanced resectable ESCC were evaluated using whole-exome sequencing (WES), whole-transcriptome sequencing and spatially resolved digital spatial profiling (DSP). To validate the findings, immunohistochemistry was conducted and a single-cell transcriptomic dataset was analysed. RESULTS: WES revealed 15.72%, 5.02% and 32.00% unique mutations in PTsup , PTdeep and LNmet , respectively. Copy number alterations and phylogenetic trees showed spatial ITH among subregions both within and among patients. Driver mutations had a mixed intra-tumoral clonal status among subregions. Transcriptome data showed distinct differentially expressed genes among subregions. LNmet exhibited elevated expression of immunomodulatory genes and enriched immune cells, particularly when compared with PTsup (all P < .05). DSP revealed orthogonal support of bulk transcriptome results, with differences in protein and immune cell abundance between subregions in a spatial context. The integrative analysis of multi-omics data revealed complex heterogeneity in mRNA/protein levels and immune cell abundance within each subregion. CONCLUSIONS: This study comprehensively characterised spatial ITH in ESCC, and the findings highlight the clinical significance of unbiased molecular classification based on multi-omics data and their potential to improve the understanding and management of ESCC. The current practices for tissue sampling are insufficient for guiding precision medicine for ESCC, and routine profiling of PTdeep and/or LNmet should be systematically performed to obtain a more comprehensive understanding of ESCC and better inform treatment decisions.

CNOT4 suppresses nonsmall cell lung cancer progression by promoting the degradation of PAF1.

CCR4-NOT transcription complex subunit 4 (CNOT4) and RNA polymerase II-associated factor, homolog (Saccharomyces cerevisiae) (PAF1) are implicated in nonsmall cell lung cancer (NSCLC). However, the molecular mechanism of their interaction in NSCLC progression is unknown. The expression of PAF1 and CNOT4 in human NSCLC tissues was detected by quantitative polymerase chain reaction. A549 cells that stably expressed CNOT4 and/or PAF1 were established. Western blot analysis and co-immunoprecipitation experiments were performed to reveal the interaction between CNOT4 and PAF1. Proliferation, migration, epithelial-mesenchymal transition (EMT), and colony formation assays were performed to determine the effect of CNOT4-PAF1 axis on NSCLC metastasis and stemness. Xenograft mouse tumor model was established, and tumor progression, EMT, and stemness were evaluated. It was found that CNOT4 expression was downregulated, whereas PAF1 expression was upregulated in human NSCLC tissues. CNOT4 facilitated the ubiquitination and degradation of PAF1 via the 26S proteasome. CNOT4 overexpression inhibited NSCLC progression, whereas PAF1 overexpression enhanced the proliferation, migration, and stemness of NSCLC, both in vitro and in vivo. Our results suggest that CNOT4-PAF1 axis modulates NSCLC metastasis and stemness, and may serve as potential therapeutic targets for lung cancer treatment.

Circlehunter: a tool to identify extrachromosomal circular DNA from ATAC-Seq data.

In cancer, extrachromosomal circular DNA (ecDNA), or megabase-pair amplified circular DNA, plays an essential role in intercellular heterogeneity and tumor cell revolution because of its non-Mendelian inheritance. We developed circlehunter ( https://github.com/suda-huanglab/circlehunter ), a tool for identifying ecDNA from ATAC-Seq data using the enhanced chromatin accessibility of ecDNA. Using simulated data, we showed that circlehunter has an F1 score of 0.93 at 30× local depth and read lengths as short as 35 bp. Based on 1312 ecDNAs predicted from 94 publicly available datasets of ATAC-Seq assays, we found 37 oncogenes contained in these ecDNAs with amplification characteristics. In small cell lung cancer cell lines, ecDNA containing MYC leads to amplification of MYC and cis-regulates the expression of NEUROD1, resulting in an expression pattern consistent with the NEUROD1 high expression subtype and sensitive to Aurora kinase inhibitors. This showcases that circlehunter could serve as a valuable pipeline for the investigation of tumorigenesis.

Effects of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) targeted combined chemotherapy on immune function, tumor markers and oxidative stress in patients with stage IV lung adenocarcinoma.

Objective: To investigate the effects of EGFR-TKI combined chemotherapy on immune function, tumor markers and oxidative stress in patients with stage-IV lung adenocarcinoma. Methods: This retrospective observational study included 116 patients with stage-IV lung adenocarcinoma, treated in The First Affiliated Hospital of Soochow University from January 2021 to January 2022. According to the treatment records, 60 patients that received pemetrexed + cisplatin for four courses were set as a Control-group and 56 patients that received EGFR-TKI + pemetrexed + cisplatin for four courses were set as an Observation-group. Changes in immune function, tumor marker levels and oxidative stress level in the two groups were analyzed and compared. Results: After the treatment, levels of CD3+, CD4+, IgG and IgM in the Control-group were significantly lower than those before the treatment. EGFR-TKI + pemetrexed + cisplatin resulted in levels of CD3+, CD4+, IgG and IgM higher than before the treatment, and compared to the Control-group (p<0.001). After the treatment, the levels of NSE, serum CEA, serum CA125, CYFEA21-1 in both groups were significantly lower than those before treatment, and lower in the Observation-group (p<0.001). After the treatment, VEGF and MMP9 levels in both groups were significantly lower than those before treatment, and markedly lower in the Observation-group (p<0.001). Conclusion: Compared with systemic chemotherapy, EGFR-TKI targeted combined chemotherapy for stage-IV lung adenocarcinoma is associated with enhanced immune function of patients. It more effectively inhibits the growth and proliferation of tumor cells and reduces the level of oxidative stress.

Exosomal LOC85009 inhibits docetaxel resistance in lung adenocarcinoma through regulating ATG5-induced autophagy.

AIMS: This study aims at investigating the role of a neighbor long non-coding RNA (lncRNA) of HDAC4 (LOC85009) in docetaxel (DTX) resistance of lung adenocarcinoma (LUAD). METHODS: RT-qPCR was used to analyze LOC85009 expression in DTX-resistant LUAD cells. In vitro and in vivo experiments were applied to detect the influence of LOC85009 on LUAD cell growth and xenograft tumor growth. DNA pull down assay, RNA pull down assay, ChIP assay, CoIP assay and RIP assay were performed to identify the direct interactions between factors. RESULTS: LOC85009 was lowly-expressed in DTX-resistant LUAD cells. Functionally, LOC85009 overexpression inhibited DTX resistance and cell proliferation but triggered cell apoptosis. Moreover, we identified that LOC85009 was transferred from LUAD cells to DTX-resistant LUAD cells via exosomes. Exosomal LOC85009 inhibited DTX resistance, proliferation and autophagy while induced apoptosis in DTX-resistant cells. Additionally, we found that LOC85009 sequestered ubiquitin-specific proteinase 5 (USP5) to destabilize upstream transcription factor 1 (USF1) protein, thereby inactivating ATG5 transcription. CONCLUSIONS: Exosomal LOC85009 inhibits DTX resistance through regulation of ATG5-induced autophagy via USP5/USF1 axis, suggesting that LOC85009 might be a potential target to reverse DTX resistance in the treatment of LUAD.

Clinical analysis of subxiphoid vs. lateral approaches for treating early anterior mediastinal thymoma.

Objective: To investigate the clinical efficacy of the subxiphoid approach for early anterior mediastinal thymoma and evaluate its advantages over the lateral intercostal approach. Methods: A total of 345 patients with early anterior mediastinal thymoma were retrospectively analyzed from January 2016 to December 2020 in the First Affiliated Hospital of Soochow University. Out of these, 99 patients underwent subxiphoid video-assisted thoracoscopic thymectomy and 246 patients underwent transthoracic video-assisted thoracoscopic thymectomy. We compared the intraoperative conditions (such as operation time and intraoperative blood loss), postoperative conditions [such as postoperative pleural drainage volume, extubation time, postoperative hospital stay, and postoperative visual analogue scale (VAS) pain score], and postoperative complications (such as death, pneumonia, delayed wound healing, cardiac arrhythmia, and phrenic nerve injury) of the two groups and analyzed the clinical advantages of the subxiphoid approach for treating early anterior mediastinal thymoma. Results: There was no significant difference between the two groups in terms of general clinical features, operation time, and postoperative complications (P > 0.05).However, there was a significant difference in terms of intraoperative blood loss, postoperative pleural drainage volume, tube extubation time, postoperative hospital stay, postoperative VAS pain score, and postoperative analgesics (a significantly decreased flurbiprofen axetil amount) (P < 0.05). Conclusion: Compared with the lateral intercostal thoracic approach, the subxiphoid approach had advantages in terms of intraoperative blood loss, postoperative hospital stay, tube extubation time, postoperative pleural drainage volume, postoperative VAS pain score, and analgesics dosage. It could provide a better view of the bilateral pleural cavities and more thorough thymectomy and superior cosmesis, and it proved to be a safe and feasible minimally invasive surgical method.

Effect of balanced opioid-free anaesthesia on postoperative nausea and vomiting after video-assisted thoracoscopic lung resection: protocol for a randomised controlled trial.

INTRODUCTION: Opioid-free anaesthesia (OFA) may reduce opioid-related side effects such as postoperative nausea and vomiting (PONV) and hyperalgesia. This study aims to investigate the effects of balanced OFA on PONV and pain outcomes in patients undergoing video-assisted thoracoscopic surgery (VATS). METHODS AND ANALYSIS: This randomised controlled trial will be conducted at the First Affiliated Hospital of Soochow University in Suzhou, China. A total of 120 adults scheduled for VATS lung resection will be randomly assigned with a 1:1 ratio to either an OFA group or a control group, stratified by sex (n=60 in each group). Patients will receive balanced anaesthesia with esketamine, dexmedetomidine and sevoflurane (the OFA group), or sufentanil and sevoflurane (the control group). All patients will receive PONV prophylaxis with intraoperative dexamethasone and ondansetron. Multimodal analgesia consists of intraoperative flurbiprofen axetil, ropivacaine infiltration at the end of surgery and postoperative patient-controlled sufentanil. The primary outcome is the incidence of PONV within 48 hours after surgery. Secondary outcomes are nausea, vomiting, need for antiemetic therapy, pain scores at rest and while coughing, postoperative sufentanil consumption, need for rescue analgesia, length of post-anaesthesia care unit stay, length of postoperative hospital stay, and 30-day and 90-day post-surgical pain and mortality. Safety outcomes are hypotension, bradycardia, hypertension, tachycardia, interventions for haemodynamic events, level of sedation, headache, dizziness, nightmare and hallucination. All analyses will be performed in the modified intention-to-treat population. ETHICS AND DISSEMINATION: Ethics approval was obtained from the Ethics Committee of the First Affiliated Hospital of Soochow University (2022-042). All patients will provide written informed consent. The results of this study will be published in a peer-reviewed journal. TRIAL REGISTRATION NUMBER: Chinese Clinical Trial Registry (ChiCTR2200059710).

Establishment and validation of an individualized nomogram for survival prediction of primary mediastinal germ cell tumors based on the SEER database.

Background: Primary mediastinal germ cell tumors (PMGCT) represent a rare but sometimes highly aggressive type of mediastinal tumors. The current prognostic models for PMGCT are insufficient. This study was undertaken to establish and validate an individualized nomogram for predicting the overall survival (OS) of patients with PMGCT. Methods: We conducted a retrospective analysis of patients with PMGCT diagnosed between 2000 and 2018 in the Surveillance, Epidemiology, and End Results (SEER) database in the United States. Clinical variables included surgery subtype, gender, treatment regimens, age, histology, tumor size, stage, chemotherapy, radiation, race, and survival-related information. The main outcome measure was survival duration. The Kaplan-Meier method along with the log-rank test were utilized to estimate the OS. Independent prognostic factors were identified by performing the univariate and multivariate Cox proportional hazards regression analyses, from which an individualized nomogram was constructed to predict 3-, 5-, and 10-year OS of patients with PMGCT. The concordance index (C-index) and calibration curve were used to verify the discrimination and accuracy of the nomogram. Results: A total of 845 patients with PMGCT were recruited from the SEER database and further randomly assigned to a training set (n=635) and a validation set (n=210) at a ratio of 7:3. The 3-, 5-, and 10-year OS for overall PMGCT was 70.0%, 67.1%, and 63.9%, respectively. Cox regression analysis indicated that age, tumor size, stage, chemotherapy, radiation, histology, and surgery type were as independent factors for OS in patients with PMGCT (P<0.05). An individualized nomogram for OS was constructed utilizing these variables, with the C-index of 0.714 [95% confidence interval (CI): 0.695 to 0.743] and 0.756 (95% CI: 0.735 to 0.787) in the training and validation groups. Moreover, good levels of agreement were observed according to the calibration curve between the predicted and actual 3-, 5-, and 10-year survival rates both in the training and validated cohorts, showing that the model could accurately predict patient prognosis. Conclusions: This study documented the first attempt at establishing and validating a novel nomogram for predicting the 3-, 5-, and 10-year OS probabilities of PMGCT. The prognostic nomogram was demonstrated to have good performance for predicting individualized OS of patients with PMGCT.

MiR-504-3p Has Tumor-Suppressing Activity and Decreases IFITM1 Expression in Non-Small Cell Lung Cancer Cells.

Objective: To analyze the impact of expression of miR-504-3p on the proliferation, migration, cell cycle transit and rate of apoptosis of NSCLC cells and explore the underlying mechanisms. Methods: The Cancer Genome Atlas (TCGA) database was used to compare the expression levels of miR-504 between NSCLC tissues and normal lung tissues. NSCLC cells were transfected with lentiviral vectors that either overexpressed or knocked down miR-504-3p to evaluate its effects on NSCLC biological behavior. Quantitative Real Time Polymerase Chain Reaction was used to measure the levels of miR-504-3p and Interferon-Induced Transmembrane Protein 1 (IFITM1). A luciferase reporter array was used to reveal whether miR-504-3p directly targets IFITM1. Results: The expression of miR-504 was significantly down-regulated in lung cancer tissues compared to normal lung tissues. Overexpression of miR-504-3p in NSCLC cell lines inhibited cell proliferation, migration and promoted cell apoptosis. Meanwhile, changes in the expression level of miR-504-3p had no significant effect on NSCLC cell cycle progression. Moreover, over-expressed miR-504-3p following its transfection significantly decreased the expression of IFITM1 in NSCLC cell lines and suppressed the activity of the luciferase reporter containing wild type but not mutant IFITM1 3' -UTR. Conclusion: miR-504-3p inhibits cell proliferation and migration and promotes cell apoptosis in NSCLC cells. MiR-504-3p decreases IFITM1 expression in NSCLC cells, which may be a potential mechanism of its tumor-suppressive functions in NSCLC.

Clinical application of preserving spontaneous breathing non-intubation anesthesia in thoracoscopic surgery for lung cancer under ERAS concept.

OBJECTIVE: To explore the safety and feasibility of thoracoscopic surgery in patients with lung cancer under non-intubation anesthesia, and to evaluate the advantages of the non-intubation anesthesia compared with intubation anesthesia on enhanced recovery after surgery (ERAS). METHODS: A retrospective cohort study was conducted in which 100 patients who underwent thoracoscopic lung cancer surgery from January 2020 to February 2021 in the Department of Thoracic Surgery of the First Affiliated Hospital of Soochow University were included and divided into non-intubation group (n = 50) and intubation group (n = 50). The primary outcome was the comparison of intra- and postoperative parameters. Secondary outcomes included inflammatory response indicators and intra- and postoperative complications. RESULTS: There was no significant difference between the two groups in anesthesia effect score, blood loss, lowest pulse oxygen saturation, operation time, postoperative chest tube indwelling time (P > 0.05). Non-intubation group had less intraoperative remifentanil dosage, less change of blood pressure and heart rate, lower postoperative pain numerical score(NRS), less medical costs, smaller incidence rate of throat discomfort (P < 0.05). The non-intubation group was also associated with less extubation time, postanesthesia care unit recovery time, ambulation time, food intake time, postoperative antibiotic use time, and hospital stay (P < 0.05). The increase of C-reactive protein in the non-intubation group was lower than that in the intubation group (P < 0.05). CONCLUSION: Non-intubation anesthesia for thoracoscopic lung cancer surgery is safe and feasible. Compared with the intubation anesthesia, it has advantages in ERAS and reducing medical costs.

The role of seven autoantibodies in lung cancer diagnosis.

BACKGROUND: To investigate the expression and diagnostic value of seven autoantibodies (P53, PGP9.5, SOX2, GAGE7, GBU4-5, MAGE, and CACE) in lung cancer patients. METHODS: A total of 370 patients were admitted to the Thoracic Surgery of the First Affiliated Hospital of Suzhou University from 2017 to 2019, including 305 patients with lung cancer and 65 patients with benign lesions. The concentrations of the seven autoantibodies were determined by enzyme linked immunosorbent assay (ELISA).The expression levels of each antibody were compared between the two groups, and the levels of each antibody between lung cancer patients with different pathological types were also compared. We aimed to analyze the diagnostic efficiency of single antibody detection combined with seven antibodies, and also to explore whether there were differences among the positive rates of each antibody in sex, age, smoking history, pathological classification, and clinical stages in the lung cancer group. RESULTS: The expression levels of seven autoantibodies in the lung cancer group were higher than those in the benign lesion group. In the lung cancer group, the expression levels of the seven autoantibodies did not vary statistically among different pathological types. The area under the curve of combined detection of the seven antibodies reached 0.735, and the Y-index reached 0.35, which was higher than that of single antibody detection. P53 exhibited the highest sensitivity and lowest specificity; meanwhile, PGP9.5, SOX2, GAGE7, GBU4-5, and MAGEA1 exhibited low sensitivity and high specificity. The sensitivity and specificity of the CAGE were approximately 60%, respectively. There was no statistical difference in the positive rate of each antibody in age, smoking history, and clinical stage. The positive rate of MAGEA1 and CAGE was statistically different in sex, and the positive rate of MAGEA1 was statistically different in pathological classification. CONCLUSIONS: The seven autoantibodies of lung cancer can potentially be used as an auxiliary examination method for the early diagnosis of lung cancer.

FAM196B promotes proliferation and migration via regulating epithelial-mesenchymal transition in esophageal cancer.

BACKGROUND: EC (esophageal cancer) is a common cancer among people in the world. The molecular mechanism of FAM196B (family with sequence similarity 196 member B) in EC is still unclear. This article aimed to clarify the role of FAM196B in EC. METHODS: The expression of FAM196B in EC tissues was detected using qRT-PCR. The prognosis of FAM196B in EC patients was determined by log-rank kaplan-Meier survival analysis and Cox regression analysis. Furthermore, shRNA was used to knockdown the expression of FAM196B in EC cell lines. MTT, wound healing assays and western blot were used to determine the role of FAM196B in EC cells. RESULTS: In our research, we found that the expression of FAM196B was up-regulated in EC tissues. The increased expression of FAM196B was significantly correlated with differentiation, lymph node metastasis, stage, and poor survival. The proliferation and migration of EC cells were inhibited after FAM196B-shRNA transfection in vitro and vivo. The western blot result showed that FAM196B could regulate EMT. CONCLUSION: These results suggested that FAM196B severs as an oncogene and promotes cell proliferation and migration in EC. In addition, FAM196B may be a potential therapeutic target for EC patients.

RPB5-mediating protein promotes the progression of non-small cell lung cancer by regulating the proliferation and invasion.

BACKGROUND: This study aimed to investigate the relationship between RNA polymerase II subunit 5 (RPB5)-mediating protein (RMP) and clinicopathological characteristics of non-small cell lung cancer (NSCLC) patients by measuring the expression level of RMP in human NSCLC tissues and cell lines. At the same time, we studied the impact of RMP on the biological function of cancer, providing strong support for gene targeted therapy of NSCLC. METHODS: Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were used to determine the expression levels of messenger (m)RNA and protein in NSCLC cell lines and tissues. Cell counting kit 8 (CCK8) assay and flow cytometry were selected to detect cell proliferation, cycle and apoptosis. The wound healing assay was chosen to detect the migration and invasion ability of cells. The xenograft model was performed to study the function of RMP in vivo. Immunohistochemical (IHC) staining showed the levels of RMP, Bcl-2, Bax and caspase-3. RESULTS: First, mRNA and protein levels of RMP were relatively overexpressed in NSCLC cells. Compared with the corresponding normal tissues, the mRNA and protein levels of RMP were significantly higher in human NSCLC tissues. Concurrently, we found that the expression of RMP was related to the status of lymph nodes (LNs) in cancer tissues and T stage. Then, RMP overexpression promoted the proliferation of A549. At the same time, RMP provided A549 cells the ability to resist chemotherapy and radiotherapy; when A549 cells were treated with gefitinib and radiation, RMP reduced apoptosis. We also found that RMP can protect A549 from G2 block caused by radiation. Over-irradiated RMP-overexpressed A549 cells had lower Bcl2-associated X protein (Bax) levels and higher B-cell lymphoma 2 (Bcl-2) levels. The migration and invasion ability of A549 cells was increased by RMP. Finally, RMP can promote tumor growth by increasing Bcl-2 levels and decreasing Bax and caspase-3 levels in the xenograft model. CONCLUSIONS: There is potential for RMP to develop into a diagnostic and therapeutic target for NSCLC.

CNOT4 suppresses non-small cell lung cancer progression and is required for effector cytolytic T lymphocytes cell responses to lung cancer cells.

The therapeutic options of non-small cell lung cancer (NSCLC) are limited, although a combination of targeted therapy and immunotherapy is promising. To explore novel targets for immunotherapy, we explored the role of Ccr4-Not transcription complex subunit 4 (CNOT4) in NSCLC. The expression of CNOT4 in tumor tissues was determined by immunohistochemistry staining and western blotting. The cell lines that stably express CNOT4 were established in H1299 and A549 cells. Direct cell counting, MTT assay, and colony formation were used to determine the ability of cell proliferation. Cell apoptosis and cell cycle were next analyzed by PI/Annexin V staining. Cell invasion and migration were examined by transwell assays. To further explore the function of CNOT4 in cytotoxic T lymphocytes (CTLs) mediated cytotoxicity, an in vitro co-culture system of CNOT4 overexpressing and control H1299 cells with CTLs was developed. CNOT4 was down-regulated in tumor tissues compared with paired normal tissues from patients with lung cancers. CNOT4 overexpression significantly inhibited tumor cell proliferation, colony formation, cell migration, and invasion, but promoted cell apoptosis. Furthermore, overexpression of CNOT4 enhanced cytotoxicity of CTLs to H1299. CNOT4 functions as a potential tumor suppressor of NSCLC via inhibiting tumor cell function and increasing the sensitivity to CTLs.

The Diagnosis of Intrapulmonary Metastasis Multifocal Pulmonary Ground-Glass Nodules Based on Oncogenic Driver Mutation: Two Case Reports and Review of Literature.

With the increased use of low-dose computed tomography (LDCT), the detection of multifocal pulmonary ground-glass nodules (GGNs) has increased. According to the current clinical guidelines, multifocal GGNs tend to be treated as the multiple primary early-stage lung adenocarcinoma. However, studies have indicated that parts of multiple GGNs may originate from single nodules via intrapulmonary metastasis (IPM). Such IPM indicates the advanced stages even when the multiple GGNs are present as the early characteristics in pathological assessments. However, no gold standard exists for the differential diagnosis of multiple IPM GGNs. Here, we report two multifocal pulmonary GGNs cases where panel sequencing (672 driver mutation loci) showed that patient 1 (P1) shared two rare epidermal growth factor receptor (EGFR) mutations (primary L747_T751del and primary T790M) in the left upper lobe anterior segment and left lower lobe superior segment, respectively. Patient 2 (P2) shared a low-frequency human epidermal growth factor receptor 2 (HER2) mutation (primary Tyr772_Ala775dup) in two GGNs located in the apicoposterior and superior lingular segment of the left lower lobe (LLL). Oncogenic driver mutations were concordant between primary tumors and metastasis. Thus, shared low-frequency driver mutations in multiple GGNs strongly suggested that IPM existed with a high probability in these patients. Also, tumor cell spread through air spaces (STAS) was identified in pathological sections of the left upper lobe (LUL) nodule of P1, suggesting aerogenous spread may have been an effective pathway for IPM. Our report suggests that oncogenic driver mutations have prominent diagnostic value for IPM. Also, GGN IPM may occur in one lung lobe and between in different lung lobes.

CNOT4 enhances the efficacy of anti-PD-1 immunotherapy in a model of non-small cell lung cancer.

The use of immune checkpoint inhibitors that target programmed cell death-1 (PD-1) has been proposed for the treatment of advanced non-small cell lung cancer (NSCLC). However, in clinical trials, cumulative response rates to anti-PD-1 treatment were approximately 20% in patients with NSCLC. CCR4-NOT transcription complex, subunit 4 (CNOT4) is a RING finger protein with E3 ubiquitin ligase activity. We previously reported that CNOT4 may act as a tumor suppressor in NSCLC. Here, we examined whether CNOT4 can enhance the efficacy of anti-PD-1 immunotherapy in a model of NSCLC. The association of CNOT4 and overall survival was analyzed using datasets from The Cancer Genome Atlas (TCGA). Tumor models were established by subcutaneously implanting tumor cells line (A549 cell) into mice. CNOT4 was observed to be positively associated with relapse-free survival and overall survival in patients with NSCLC. CNOT4 overexpression suppressed tumor growth in vivo and enhanced the effect of anti-PD-1 immunotherapy, which was accompanied by increased CD3+ and CD8+ T lymphocyte infiltration and higher interferon-γ and tumor necrosis factor-α levels. In conclusion, CNOT4 may enhance the efficacy of anti-PD-1 immunotherapy and may have potential as a prognostic marker for NSCLC, or as a combinational target with anti-PD-1 treatment for patients with NSCLC.

Insulin-like growth factor-1 receptor induces immunosuppression in lung cancer by upregulating B7-H4 expression through the MEK/ERK signaling pathway.

The Insulin-like growth factor-1/Insulin-like growth factor-1 receptor (IGF1/IGF1R) axis contributes to immunosuppression during tumor progression; however, the underlying mechanism remains unclear. In the present study, we found that IGF1 stimulation or IGF1R overexpression (IGF1R-OE) could upregulate the expression of B7-H4, while IGF1R inhibition downregulated B7-H4 in both A549 and SPC-A-1 lung cancer cell lines. IGF1R-OE conferred the inhibition of CD8+ T cells by cancer cells in vitro, and induction of B7-H4 expression was mediated by the activation of the MEK/ERK1/2 signaling pathway. The in vitro findings were further confirmed in vivo using a Lewis lung cancer mouse model. IGF1R-OE promoted tumor growth and inhibited tumor infiltration by CD8+ T cells in the mouse model. However, this effect was suppressed when B7-H4 was knocked down in IGF1R-OE cells. Our findings suggest that IGF1R could induce immunosuppression in lung cancer by upregulating the expression of B7-H4 through the MEK/ERK pathway. B7-H4 may therefore be a potential therapeutic target for lung cancer immunotherapy.

Clinicopathologic Features and the Prognostic Implications of Long Noncoding RNA HOTAIRM1 in Non-Small Cell Lung Cancer.

Aims: The purpose of this study was to explore the value of long noncoding RNA-HOXA transcript antisense RNA myeloid-specific 1 (LncRNA-HOTAIRM1) as a prognostic candidate for detecting non-small cell lung cancer (NSCLC). Materials and Methods: The cancer cell line encyclopedia online database was utilized to analyze HOTAIRM1 expression in different tumor cell lines and to estimate the relationship between HOTAIRM1 and clinicopathologic parameters based on the chi-square test. We compared the LncRNA-HOTAIRM1 levels in cancerous and paracancerous tissues of NSCLC patients using quantitative real-time polymerase chain reaction assays. The Kaplan-Meier method was performed to analyze overall survival (OS). Results: LncRNA-HOTAIRM1 showed varied expression levels in different malignant tumor cell lines. There was a significant association between the expression of HOTAIRM1 and histopathological differentiation, tumor size, tumor/node/metastasis (TNM) stage, and Ki-67 of NSCLC patients. In addition, the relative expression of HOTAIRM1 in NSCLC tissues was significantly higher when compared with the individual patients' matched paracancerous tissues. Patients in the group with low expression levels of HOTAIRM1 had a longer OS than those in the group with high expression levels for lung adenocarcinoma, I-II stages of NSCLC, and NSCLC with smoking history. Conclusion: Our study suggests that LncRNA-HOTAIRM1 is highly expressed in NSCLC, and is associated with a poor prognosis, higher clinicopathologic grade, and smoking. LncRNA-HOTAIRM1 is a potential diagnostic and prognostic biomarker for NSCLCs.