Exosomes derived from M1 macrophages inhibit the proliferation of the A549 and H1299 lung cancer cell lines via the miRNA-let-7b-5p-GNG5 axis.

Background: Almost all cells are capable of secreting exosomes (Exos) for intercellular communication and regulation. Therefore, Exos can be used as a natural therapeutic platform to regulate genes or deliver drugs to treat diseases. M1 macrophages inhibit tumor growth by releasing pro-inflammatory factors. This study explored the applicability of M1 macrophage exosomes (M1-Exos) as gene carriers and the effects on GNG5 protein, and further examined whether macrophage repolarization could inhibit tumor activity. Methods: M0 macrophages were polarized toward M1 using vitexin. Exos were obtained from M1 macrophages by ultra-centrifugation. The transwell non-contact co-culture system was used to co-culture M1 macrophages with HLF-α human lung epithelial cells or A549 or H1299 lung cancer cells. MTT, scratch, and transwell assays were used to detect the cell viability, migration, and invasion ability of cells in the four groups. Flow cytometry was used to detect the apoptosis rate of each group, and western blot (WB) analysis was performed to detect the change in the expression of proliferation- and apoptosis-related proteins. We screened the differentially expressed microRNAs using quantitative polymerase chain reaction technology. Luciferase reporter analysis was performed to explore the interaction between miRNA and protein. We used Xenografted A549 tumors in nude mice to study the effect of M1-Exos on tumor cell growth in vivo. Results: The results showed that, under the M1 macrophage co-culture system, lung cancer cell viability, invasion, and migration ability decreased, and the number of apoptotic cells increased, will all indicators being statistically significant (P < 0.05). The expression levels of PCNA, KI67, and Bcl-2 decreased significantly, but that of Bax increased (P < 0.05). Exosomes can have the same effect on tumor cells as M1 macrophages. Exosomes can transport miR-let-7b-5p to tumor cells, and miR-let-7b-5p can inhibit tumor cell proliferation and promote tumor cell apoptosis by regulating the GNG5 protein level. Conclusions: M1-Exos inhibit the proliferation, invasion, and metastasis of lung cancer cells through miRNA-let-7b-5p and GNG5 signaling pathways and inhibit the anti-apoptotic ability of lung cancer cells.

Detection of an EML4-ALK fusion mutation secondary to epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) therapy for lung cancer: a case report.

BACKGROUND: For epidermal growth factor receptor-mutant (EGFR-mutant) advanced non-small cell lung cancer (NSCLC) patients, EGFR-tyrosine inhibitors such as gefitinib, erlotinib, and osimertinib, are recommended as the preferred first-line treatment. Unfortunately, relevant drug resistance is often inevitable and for first and second generation EGFR-tyrosine kinase inhibitors (TKIs), drug resistance most commonly (50-60% of cases) occurs at the secondary point mutation T790M. Second-line treatments may include administering the third generation of EGFR-TKIs, such as osimertinib and almonertinib. In a few relevant studies, rearrangement of the anaplastic lymphoma kinase (ALK) gene was detected in patients with T790M mutation after drug resistance to osimertinib re-occurred following administration as a second-line treatment. The studies concluded that ALK rearrangement is a rare but critical drug resistance mechanism for osimertinib. However, to date, it remains unclear whether almonertinib also triggers the same ALK rearrangement. The current case study is the first one detailing the detection of an ALK rearrangement after almonertinib resistance in advanced EGFR-mutant NSCLC, which contributes to the limited body of literature examining ALK rearrangement as a mechanism of resistance to EGFR-TKIs in advanced EGFR-mutant NSCLC. CASE DESCRIPTION: Herein, we present a 35-year-old female patient with EGFR-mutant advanced NSCLC in the last trimester of pregnancy. The patient was administered multiple treatments, including first-line icotinib and second-line almonertinib. According to the next-generation sequencing (NGS) assay after almonertinib resistance, the development of an EML4-ALK fusion mutation was considered to be a potential mechanism of almonertinib resistance. Subsequently, the patient received a combination of almonertinib and crizotinib, and at the last follow-up, the treatment showed a curative effect and then maintained a one-month stable disease. CONCLUSIONS: This case report suggests that ALK rearrangement may be a potential mechanism of almonertinib resistance. The combination of ALK TKI therapy and EGFR TKI may be a viable strategy for almonertinib-resistant NSCLC patients induced by ALK rearrangement.

A systematic review and meta-analysis of circulating cell-free DNA as a diagnostic biomarker for non-small cell lung cancer.

Background: Early diagnosis of non-small cell lung cancer (NSCLC) is crucial for treatment. Circulating cell-free DNA (cfDNA) is an extracellular nucleic acid found in serum, and tumor cfDNA circulating in the blood may be used as a biomarker for early diagnosis. The purpose of this study was to evaluate the application value of cfDNA as a biomarker for the diagnosis of NSCLC through meta-analysis. Methods: We searched the China National Knowledge Infrastructure (CNKI), Wanfang, VIP, PubMed, Cochrane Central Register of Controlled Trials, Embase, and Web of Science databases using the following search terms: lung cancer, NSCLC, biomarkers, circulating cfDNA, cfDNA, circulating tumor DNA (ctDNA), circulating cell-free tumor DNA, and diagnosis. The retrieval period was set until September 2021. According to PICOS (patients, intervention, comparison, outcomes, and study design) principles the inclusion criteria were: aged ≥18 years; at least 10 NSCLC cases; NSCLC patients diagnosed by histopathology or cytology; circulating cfDNA was detected; outcome data could be completely extracted. Bias risk assessment was conducted according to the QUADAS (Quality Assessment of Diagnostic Accuracy Studies). RevMan 5.3 was used for meta-analysis. Results: Eight studies met the inclusion criteria, including a total of 618 NSCLC patients and 635 healthy subjects. The overall sensitivity and specificity were 0.79 [95% confidence interval (CI): 0.75-0.82] and 0.81 (95% CI: 0.78-0.84), respectively. The area under the curve (AUC) of the summary receiving operating characteristic (SROC) curve was 0.8941. The pooled positive likelihood ratio, pooled negative likelihood ratio, and pooled diagnostic odds ratio were 5.37 (95% CI: 2.67-10.81), 0.24 (95% CI: 0.15-0.38), and 24.68 (95% CI: 8.85-68.84), respectively. The patient selection bias was high in two articles was high, unclear in one article, and low in the remaining five ones. The risk of bias in the research index test was unclear in one article, and low in the remaining seven articles. The reference standard bias, and flow and time bias of all articles was low. Conclusions: Circulating cfDNA is an efficacy biomarker in diagnosis of NSCLC. Its clinical application technology is worthy of further research.

Efficacy and Safety of Gefitinib Plus Anlotinib for Patients with EGFR Positive Advanced Non-Small-Cell Lung Cancer: A Retrospective Exploratory Study.

Objective: This study was to investigate the efficacy and safety of gefitinib plus anlotinib for patients with EGFR positive advanced non-small cell lung cancer (NSCLC) in a first-line setting. Methods: A total of 36 patients with previously-untreated EGFR positive advanced NSCLC were included in this study retrospectively. All patients were administered with gefitinib plus anlotinib combination therapy. The efficacy of the patients was evaluated with the change of target lesion using imaging evidence according to RECIST 1.1 criteria and all the patients were followed up regularly. Adverse reactions were collected and documented during the combination administration. Univariate analysis according to the baseline characteristic subgroup was implemented using Log rank test and multivariate analysis was adjusted by Cox regression analysis. Results: All the 36 patients included in our study were available for efficacy and safety analysis. Best overall response of the patients during gefitinib plus anlotinib administration suggested that partial response was observed in 30 patients, stable disease was noted in five patients, and progressive disease was found in one patient, which yielded an objective response rate (ORR) of 83.3% (95% CI=67.2-93.6%) and a disease control rate (DCR) of 97.2% (95% CI=85.5-99.9%). Prognostic data indicated that the median progression-free survival (PFS) of the 36 patients with NSCLC was 15.2 months (95% CI=8.15-22.26). Furthermore, the median overall survival (OS) of the 36 patients was 35.9 months (95% CI=22.77-49.03). Additionally, the most common adverse reactions of the patients with NSCLC were diarrhea (63.9%), fatigue (58.3%), hypertension (50.0%), rash (44.4%), and nausea and vomiting (41.7%). Furthermore, ECOG performance status was associated with PFS of gefitinib plus anlotinib combination therapy in baseline characteristic subgroup analysis. Conclusion: Gefitinib plus anlotinib regimen demonstrated encouraging efficacy and an acceptable safety profile for patients with previously untreated EGFR positive NSCLC preliminarily. The conclusion should be validated in prospective clinical trials subsequently.

Clinical observation of crizotinib in the treatment of ALK-positive advanced non-small cell lung cancer.

BACKGROUND: ALK is a prognostic and predictive tumor marker in non-small cell lung carcinoma (NSCLC), and is more often found in lung adenocarcinomas. METHODS: The clinical and pathological data of 87 patients confirmed to have NSCLC by pathology or cytology were selected from April 2014 to January 2017 at the Tumor Hospital of Hebei Province. RESULTS: Of the 87 ALK-positive-patients, 47 patients were treated with oral administration of crizotinib. The objective response rate (ORR) was 61.7%, the disease control rate (DCR) was 93.6%, and the mPFS was 19 months. In an analysis of the number of metastatic sites, the patients who had more than three metastatic sites, the ORR, DCR, and mPFS were 63.9%, 94.5%, and 19 months, compared with the 45.5%, 91%, and 11 months in the patients with less sites (P = 0.040). For patients of 60 years or older, ORR and DCR were 40% and 100%, the other group was 71.9% and 90.6%, respectively(P = 0.036). The timing of treatment was analyzed. At the first application of crizotinib, ORR and DCR were 78.2% and 100% corresponding 45.8% and 87.5% at the second and final application of crizotinib group (P = 0.022). Baseline brain metastases were present in 25.5% (12/47) of patients in this study. 9 of the patients who developed disease progression during crizotinib treatment had new brain metastases or increased preexisting cranial foci. Most of them took the treatment strategy of continuing crizotinib or replacing the second/third generation ALK-TKI treatment combined with local radiotherapy for brain metastases. CONCLUSIONS: The efficacy of crizotinib in patients with advanced NSCLC is related to the number of metastatic organs, age and timing of treatment. The use of crizotinib is prone to intracranial progression, and progression of simple brain metastases is not an indication that crizotinib is discontinued. Patients will continue to benefit from combination of local radiotherapy.

MicroRNA­145 inhibits migration and induces apoptosis in human non­small cell lung cancer cells through regulation of the EGFR/PI3K/AKT signaling pathway.

In the present study, the therapeutic effects and the underlying molecular mechanisms of microRNA (miR)­145 were investigated in non­small cell lung cancer (NSCLC) cells. Reverse transcription­quantitative polymerase chain reaction (RT­qPCR) was performed to examine miR­145 expression. An MTT assay and flow cytometry were used to investigate cell proliferation and apoptosis, respectively. The protein expression of Bax, epidermal growth factor receptor (EGFR), phosphatidylinositol 3­kinase (PI3K) and phosphorylated­protein kinase B (AKT) was examined by western blot analysis. miR­145 expression was downregulated in patients with NSCLC who were treated with chemotherapy. The downregulation of miR­145 in A549 cells reduced lactate dehydrogenase (LDH) expression, apoptosis, caspase­3/-9 levels and Bax protein expression, while it increased cell proliferation. Upregulation of miR­145 in A459 cells increased LDH, apoptosis, caspase­3/-9 levels and Bax protein expression, while it inhibited cell proliferation. The EGFR/PI3K/AKT signaling pathway was suppressed by miR­145 upregulation in A549 cells and induced by miR­145 downregulation. The EGFR inhibitor suppressed the EGFR/PI3K/AKT signaling pathway and increased the anticancer effects of miR­145 upregulation in A549 cells. The PI3K inhibitor suppressed the PI3K/AKT signaling pathway and reversed the anticancer effects of miR­145 upregulation in A549 cells. In conclusion, the present study demonstrated that miR­145 regulates the EGFR/PI3K/AKT signaling pathway in patients with NSCLC.

Over-regulation of microRNA-133b inhibits cell proliferation of cisplatin-induced non-small cell lung cancer cells through PI3K/Akt and JAK2/STAT3 signaling pathway by targeting EGFR.

The present study determined the anticancer activity and its mechanism of microRNA­133b on cell proliferation of cisplatin-induced non-small cell lung cancer cells. The expression of microRNA-133b cisplatin­induced non-small cell lung cancer (NSCLC) tissue was lower than that of para-carcinoma tissue in patients. Overall survival of higher expression in cisplatin-induced NSCLC patients was higher than that of lower expression in cisplatin­induced NSCLC patients. Over-regulation of microRNA-133b inhibited cell proliferation and LDH activity, induced apoptosis and caspase-3 activity, suppressed the protein expression of EGFR, PI3K, p-Akt, p-JAK2 and p-STAT3, decreased cyclin D1 and increased Bax protein expression in cisplatin­induced A549 cells. EGFR inhibitor (lapatinib) suppressed EGFR protein expression, inhibited cell proliferation and LDH activity, and induced apoptosis and caspase-3 activity in cisplatin-induced A549 cells by over-regulation of microRNA-133b. When EGFR protein expression was suppressed, PI3K, p-Akt, p-JAK2 and p-STAT3, decreased cyclin D1 and increased Bax protein expression in cisplatin-induced A549 cells by over-regulation of microRNA-133b. Altogether, our results indicated that over-regulation of microRNA-133b inhibits cell proliferation of cisplatin-induced NSCLC by PI3K/Akt and JAK2/STAT3 signaling pathway by targeting EGFR.

A polymorphism at the miR-502 binding site in the 3' untranslated region of the SET8 gene is associated with the outcome of small-cell lung cancer.

microRNAs (miRNAs) bind to the 3' untranslated regions (UTRs) of messenger RNAs, where they interfere with translation of genes that regulate cell differentiation, apoptosis and tumourigenesis. The histone methyltransferase SET8 has been reported to methylate TP53 and regulate genomic stability. We analysed a single nucleotide polymorphism (rs16917496) within the miR-502 miRNA seed region at the 3' UTR of SET8 in small-cell lung cancer (SCLC) patients. The SET8 CC+CT genotype was identified to be independently associated with longer survival in SCLC patients by multivariate analysis (relative risk, 0.453; 95% CI 0.217-0.944; p=0.035). The analysis of genetic polymorphisms in miRNA binding sites may help to identify patient subgroups at high risk of poor outcome.