A novel synthesized prodrug of gemcitabine based on oxygen-free radical sensitivity inhibited the growth of lung cancer cells

In the present study,we introduced the H2O2-sensitive thiazolidinone moiety at the 4th amino group of gemcitabine(GEM)to synthesize a new target compound named GEM-ZZQ,and then we confirmed its chemical structure by nuclear magnetic resonance spectroscopy.We further confirmed that GEM-ZZQ had a good chemical stability in different pH solutions in vitro and that it could be activated by H2O2 to release GEM.Pharmacodynamic studies revealed that the growth inhibition of human normal epithelial cells was weaker by GEM-ZZQ than by GEM treatment and that the inhibition of various lung cancer cell lines by GEM-ZZQ was similar to that of GEM.For the lung cancer cell lines that are resistant to the epidermal growth factor receptor(EGFR)-targeting inhibitor osimertinib,GEM-ZZQ showed less growth inhibition than GEM;however,GEM-ZZQ in combination with cisplatin showed better synergistic effects than GEM in the low-dose groups.In summary,we provided a new anti-cancer compound GEM-ZZQ for treating lung cancer by modifying the GEM structure.

Biological insights in non-small cell lung cancer

Lung oncogenesis relies on intracellular cysteine to overcome oxidative stress.Several tumor types,including non-small cell lung cancer(NSCLC),upregulate the system x-c cystine/glutamate antiporter(xCT)through overexpression of the cystine transporter SLC7A11,thus sustaining intracellular cysteine levels to support glutathione synthesis.Nuclear factor erythroid 2-related factor 2(NRF2)serves as a master regulator of oxidative stress resistance by regulating SLC7A11,whereas Kelch-like ECH-associated protein(KEAP1)acts as a cytoplasmic repressor of the oxidative responsive transcription factor NRF2.Mutations in KEAP1/NRF2 and p53 induce SLC7A11 activation in NSCLC.Extracellular cystine is crucial in supplying the intracellular cysteine levels necessary to combat oxidative stress.Disruptions in cystine availability lead to iron-dependent lipid peroxidation,thus resulting in a type of cell death called ferroptosis.Pharmacologic inhibitors of xCT(either SLC7A11 or GPX4)induce ferroptosis of NSCLC cells and other tumor types.When cystine uptake is impaired,the intracellular cysteine pool can be sustained by the transsulfuration pathway,which is catalyzed by cystathionine-B-synthase(CBS)and cystathionine g-lyase(CSE).The involvement of exogenous cysteine/cystine and the transsulfuration pathway in the cysteine pool and downstream metabolites results in compromised CD8^(+)T cell function and evasion of immunotherapy,diminishing immune response and potentially reducing the effectiveness of immunotherapeutic interventions.Pyroptosis is a previously unrecognized form of regulated cell death.In NSCLCs driven by EGFR,ALK,or KRAS,selective inhibitors induce pyroptotic cell death as well as apoptosis.After targeted therapy,the mitochondrial intrinsic apoptotic pathway is activated,thus leading to the cleavage and activation of caspase-3.Consequently,gasdermin E is activated,thus leading to permeabilization of the cytoplasmic membrane and cell-lytic pyroptosis(indicated by characteristic cell membrane ballooning).Breakthroughs in KRAS G12C allele-specific inhibitors and potential mechanisms of resistance are also discussed herein.

Prevalence and clinical significance of pathogenic germline BRCA1/2 mutations in Chinese non-small cell lung cancer patients

Objective: Germline alterations in the breast cancer susceptibility genes type 1 and 2, BRCA1 and BRCA2, predispose individuals to hereditary cancers, including breast, ovarian, prostate, pancreatic, and stomach cancers.Accumulating evidence suggests inherited genetic susceptibility to lung cancer.The present study aimed to survey the prevalence of pathogenic germline BRCA mutations(gBRCAm) and explore the potential association between gBRCAm and disease onset in Chinese advanced non-small cell lung cancer(NSCLC) patients.Methods: A total of 6,220 NSCLC patients were screened using capture-based ultra-deep targeted sequencing to identify patients harboring germline BRCA1/2 mutations.Results: Out of the 6,220 patients screened, 1.03%(64/6,220) of the patients harbored the pathogenic gB RCAm, with BRCA2 mutations being the most predominant mutations(49/64, 76.5%).Patients who developed NSCLC before 50 years of age were more likely to carry gBRCAm(P = 0.036).Among the patients harboring classic lung cancer driver mutations, those with concurrent gBRCAm were significantly younger than those harboring the wild-type gBRCA(P = 0.029).By contrast, the age of patients with or without concurrent gBRCAm was comparable to those of patients without the driver mutations(P = 0.972).In addition, we identified EGFR-mutant patients with concurrent gBRCAm who showed comparable progression-free survival but significantly longer overall survival(P = 0.002) compared to EGFR-mutant patients with wild-type germline BRCA.Conclusions: Overall, our study is the largest survey of the prevalence of pathogenic gBRCAm in advanced Chinese NSCLC patients.Results suggested a lack of association between germline BRCA status and treatment outcome of EGFR-TKI.In addition,results showed a positive correlation between pathogenic gB RCAm and an early onset of NSCLC.

miR-124 Modulates Gefitinib Resistance through SNAI2 and STAT3 in Non-small Cell Lung Cancer

Gefitinib is used as a first-line treatment for advanced non-small cell lung cancer（NSCLC）.Unfortunately,most NSCLC patients inevitably develop gefitinib resistance during treatment.In addition to EGFR mutation status,the mechanisms involved are largely unknown.In this study,we showed that mi R-124,a tumor suppressor,was significantly down-regulated in gefitinib-resistant NSCLC patients and cell lines compared with gefitinib-sensitive patients and cell lines.In addition,the mi R-124 depletion induced gefitinib resistance,and mi R-124 overexpression sensitized gefitinib-resistant cells to gefitinib.Mechanistic analysis revealed that mi R-124 decreased SNAI2 and STAT3 expression by directly targeting their 3＇UTRs and that knocking down SNAI2 or STAT3 partly reversed the gefitinib resistance induced by mi R-124 depletion.Our data demonstrate that the mi R-124 plays a new critical role in acquired resistance to gefitinib and that the manipulation of mi R-124 might provide a therapeutic strategy for reversing acquired gefitinib resistance.

New horizons for uncommon mutations in non-small cell lung cancer: BRAF, KRAS, RET, MET, NTRK, HER2

The 2004 discovery of EGFR mutations,followed by ALK rearrangements,ushered in a targeted therapy era for advanced non-small cell lung cancer(NSCLC).Tyrosine kinase inhibitors targeting gene alterations have substantially improved survival and quality of life for patients with NSCLC.In the last decade,rearrangements of the ROS1 oncogene have been incorporated into healthcare practice that are applicable to another small subgroup of patients who benefit from similar targeted strategies.Recent genome studies of lung adenocarcinoma have identified other possible therapeutic targets,including RET,NTRK fusions,c-MET alterations,and activating mutations in KRAS,BRAF,and HER2,all with frequencies greater than 1%.Lung cancers harbouring these genome changes can potentially be treated with agents approved for other indications or under clinical development.This review updates the therapeutic arsenal that especially targets those genes.

Expression of VEGFR2 and NRP-1 in non-small cell lung cancer and their clinical significance

Objective： Vascular-targeted therapy is gradually becoming more appealing for patients with lung cancer. It is unclear whether vascular endothelial growth factor receptor 2（VEGFR2） and neuropilin-1（NRP-1） can be biomarkers for clinical treatment. We aimed to investigate the expression levels of VEGFR2 and NRP-1 in human non-small cell lung cancer（NSCLC） and their clinical significance by observing patient prognosis. Methods： VEGFR2 and NRP-1 were assessed by immunohistochemistry（IHC） in 40 patients with NSCLC and in 10 patients with benign lesions of lung; kinase insert domain receptor（KDR） and NRP-1 copy number gain（CNG） was assessed by fluorescence in situ hybridization（FISH）. The distributions of overall survival（OS） and progression-free survival（PFS） were estimated using the Kaplan-Meier method and compared between groups by log-rank test.Results： Rates of positive immunostaining for VEGFR2 and NRP-1 were 58% and 55%, respectively. KDR and NRP-1 CNG（＋） were detected in 32.5% and 30% of tumors, respectively. Levels of both VEGFR2 and NRP-1 in lung tumors were significantly different than in the control tissue（χ2=11.22, P=0.001; χ2=9.82, P=0.001, respectively）; similar results were obtained using CNGs（χ2=4.39, P=0.036; χ2=3.95, P=0.046, respectively）. Statistically significant correlations were observed with histological grade, clinical TNM stage and the lymph node status（P〈0.05）, but not age, gender or pathology type（P〉0.05）. VEGFR2 showed a strong correlation with NRP-1（Rs=0.68, P=0.00）; similar results were observed with KDR and NRP-1 CNG（Rs=0.32, P=0.04）. Significant differences in OS and PFS were observed between the groups with higher VEGFR2 and NRP-1 and those with lower expression（P〈0.05）. Conclusions： According to these data, VEGFR2 and NRP-1 are highly expressed in NSCLC. We can conclude that they play a key role in NSCLC occurrence, development and metastasis and are associated with patient prognosis（P〈0.05 for OS and PFS）. This information will be beneficial for clinical antiangiogenic treatment in NSCLC.

Clinical benefit of COX-2 inhibitors in the adjuvant chemotherapy of advanced non-small cell lung cancer: A systematic review and metaanalysis

BACKGROUND Lung cancer is a major cause of death among patients,and non-small cell lung cancer(NSCLC)accounts for more than 80%of all lung cancers in many countries.AIM To evaluate the clinical benefit(CB)of COX-2 inhibitors in patients with advanced NSCLC using systematic review.METHODS We searched the six electronic databases up until December 9,2019 for studies that examined the efficacy and safety of the addition of COX-2 inhibitors to chemotherapy for NSCLC.Overall survival(OS),progression free survival(PFS),1-year survival rate(SR),overall response rate(ORR),CB,complete response(CR),partial response(PR),stable disease(SD),and toxicities were measured with more than one outcome as their endpoints.Fixed and random effects models were used to calculate risk estimates in a meta-analysis.Potential publication bias was calculated using Egger’s linear regression test.Data analysis was performed using R software.RESULTS The COX-2 inhibitors combined with chemotherapy were not found to be more effective than chemotherapy alone in OS,progression free survival,1-year SR,CB,CR,and SD.However,there was a difference in overall response rate for patients with advanced NSCLC.In a subgroup analysis,significantly increased ORR results were found for celecoxib,rofecoxib,first-line treatment,and PR.For adverse events,the increase in COX-2 inhibitor was positively correlated with the increase in grade 3 and 4 toxicity of leukopenia,thrombocytopenia,and cardiovascular events.CONCLUSION COX-2 inhibitor combined with chemotherapy increased the total effective rate of advanced NSCLC with the possible increased risk of blood toxicity and cardiovascular events and had no effect on survival index.

A causal variant rs3769823 in 2q33.1 involved in apoptosis pathway leading to a decreased risk of non-small cell lung cancer

Objective:Although our previous genome-wide association study(GWAS)has identified chromosome 2q33.1 as a susceptibility locus for non-small cell lung cancer(NSCLC),the causal variants remain unclear.The aims of this study were to identify the causal variants in 2q33.1 and to explore their biological functions in NSCLC.Methods:CCK-8,colony formation,EdU incorporation,Transwell,and quantitative real-time polymerase chain reaction assays were applied to examine variant function.The tumor xenograft model was used to examine variant function in vivo.Caspase-8 activity assays,flow cytometry analysis,and co-immunoprecipitation assays were used to explore the molecular mechanism.Results:The missense variant rs3769823(A>G),which caused the substitution of lysine with arginine at amino acid 14 in caspase-8(caspase-8K14R),was identified as a potential causal candidate in 2q33.1.Compared with the wild type caspase-8(caspase8WT)group,the caspase-8K14R group had higher expression of caspase-8 and cleaved caspase-8.Caspase-8K14R inhibited the proliferation and metastasis of human lung cancer cell lines in vitro.Moreover,caspase-8K14R repressed lung cancer cell growth in vivo.Mechanistically,caspase-8K14R was more sensitive than caspase-8WT to tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)-mediated apoptosis and showed higher binding of caspase-8 and FADD.Conclusions:These results suggested that rs3769823 is the causal variant in chromosome 2q33.1 and is involved in an apoptosis pathway,leading to a decreased risk of NSCLC.

A Phase Ⅱ Clinical Trial of Celecoxib Combined with Platinum-Based Regimen as First-Line Chemotherapy for Advanced Non-Small Cell Lung Cancer Patients with Cyclooxygenase-2 Positive Expression

Objective： To evaluate the efficacy and safety of celecoxib treatment of advanced non-small cell lung cancer （NSCLC）, and combined therapy by molecular analysis. plus platinum-doublet as first-line chemotherapy in to determine the subgroup benefiting from celecoxib Methods： A total of 44 treatment-naive patients of advanced NSCLC with positive cyclooxygenase-2 （COX-2） expression confirmed by immunohistochemical （IHC） staining were designed to receive celecoxib plus platinum-doublet chemotherapy （cisplatin plus gemcitabine, novelbine or docetaxol） from February 2005 to May 2007. On 5-7 day before chemotherapy, 400 mg celecoxib was administered twice a day orally until obvious evidence of disease progression or intolerable toxicity was found. Adverse events were recorded according to NCI-CTC criteria. The primary endpoint was overall survival （OS）. The secondary endpoints included progression-free survival （PFS）, 1-year survival rate, response rate （RR） and safety. Additionally, we detected epithelial growth factor receptor （EGFR） status including EGFR gene amplification by real-time PCR and gene mutations by DHPLC followed by sequencing. Results： The response rate was 45% （20/44）, and the disease control rate （DCR） was 59% （26/44）. The median progression-free survival time and median survival time were 6 m and 18 m, respectively. The l-year survival rate was 68%. Chemotherapy cycle numbers and best response were found to be the predictive factors for PFS by COX model analysis （P=0.023 and P=0.000, respectively）. No factor was found to affect OS. The most common toxicities included neutropenia and nausea/vomit. EGFR gene amplification was an independent prognostic factor influencing OS （P=0.0002）. Patients with EGFR mutations （exon 21） had a tendency of disease progression （P=0.041）. Conclusion： Encouraging activities of celecoxib combined with platinum-doublet chemotherapy were demonstrated in treatment-naive patients with advanced NSCLC, with good tolerances. For COX-2 IHC positive patients, positive EGFR amplification and mutation might be related to poor clinical outcomes.

Targeting HER2 genomic alterations in non-small cell lung cancer

Oncogenic mutations and amplifications in the erythroblastic oncogene B(ERBB2),or human epidermal growth factor receptor 2(HER2),have emerged as distinct oncogenic drivers and drug targets in non-small cell lung cancer(NSCLC).Each genomic alteration occurs in 2-4%of NSCLC by next generation sequencing and is asso-ciated with constitutive HER2 activation.The most common HER2 mutations in NSCLC are exon 20 mutation A775_G776insYVMA mutation in the kinase domain and S310F mutation in the extracellular domain.Unlike in breast and gastric cancer,HER2 protein overexpression in NSCLC is not validated to be a biomarker predictive of clinical response to HER2-targeted agents.High HER2 protein overexpression by immunohistochemistry(3+)only occurs in 2-4%of NSCLC.Until now HER2-targeted agents(such as afatinib and ado-trastuzumab emtansine)only demonstrate modest clinical activity in patients with HER2-mutant NSCLC.Retrospective studies show concern for inferior clinical benefit of immune checkpoint inhibitors in HER2-mutated NSCLC.Therefore,platinum-based chemotherapy with or without an anti-angiogenesis inhibitor remains the first line standard treatment for this pa-tient population.In May 2020 trastuzumab deruxtecan(T-DXd)received the U.S.Food and Drug Administration breakthrough therapy designation for HER2-mutant metastatic NSCLC,and was added as an option for HER2-mutant NSCLC to the NCCN guidelines V1.2021.A global phase III study of pyrotinib compared to docetaxel as a second line therapy for advanced NSCLC harboring HER2 exon 20 mutations was just opened for enrollment in September 2020.In this review,we highlight the current knowledge and perspectives on targeting-HER2 genomic alterations in NSCLC.

The effects of the same target on malignant proliferation of human lung cancer cells with different expression levels of COX-2 protein

Objective: The aim of the study was to explore the effects of the same target (si-10) on lung cancer cells with different expression levels of cyclooxygenase-2 (COX-2) protein by RNAi and malignant proliferation of these cells. Methods: COX-2 was selected as the target and one siRNA expression vector with the best effect was selected and thought as the subject from three COX-2 siRNA expression vectors with human U6 promoter. The siRNA expression vector (psi-10) and the vacant vector (pEGFP) were transfected into these cells with different COX-2 expression states (801D, A549 and LTEP-A2) with lipofectamine respectively and the transfected cell strains were constructed. The change of COX-2 expression levels was examined by Western blot and RT-PCR. The effects on the proliferation of lung cancer cells were studied by cell growth curve and clonogenic assay. Results: The siRNA and U6 promoter were validated by PCR, restriction endonucleases identification and DNA sequencing and BLAST alignment and cloned into the pEGFP vector. The cell strains transfected that 801D was used as maternal line were named as 801D-p and 801D-10 respectively. The cell strains transfected that A549 was used as maternal line were named as A549-p and A549-10 respectively. The cell strains transfected that LTEP-A2 was used as maternal line were named as LTEP-A2-p and LTEP-A2-10 respectively. These cells transfected pEGFP (801D-p, A549-p and LTEP-A2-p) had the expression of GFP and 801D-10, A549-10 and LTEP-A2-10 cells had not in 24, 48 and 72 hours after transfected. The results of RT-PCR and Western blot showed the siRNA expression vector produced marked effects in two cells (A549 and LTEP-A2) expressing COX-2 and the expression of COX-2 was inhibited. But the inhibited effects were differ- ent and the expression of COX-2 was more inhibited obviously in LTEP-A2 cells than in A549 cells though the expression of COX-2 was also inhibited obviously in A549 cells. In contract to their maternal line, the levels of COX-2 mRNA of LTEP-A2-10 and A549-10 cells reduced 64.2% and 61.2% respectively; the levels of COX-2 protein reduced 60.2% and 56.2% respectively. But the levels of COX-2 mRNA and protein had not change in 801D cells not expressing COX-2. The results of cell growth curve and clonogenic assay showed the growth of LTEP-A2-10 cells slowed and the clonal formation rate reduced and the size of the colonies became small; the growth of A549-10 cells showed slow and more obviously in the cell growth curve especially. But the growth of 801D-10 cells had not obvious change. Conclusion: The si-10 target of COX-2 has different inhibition effects on lung cancer cells with different COX-2 expression levels and the different inhibition effects have different effects on cells malignant proliferation.

EGFR / HER2 / HER3 expression in tumour and gefitinib treatment in Chinese patients with advanced non-small cell lung cancer

Objective： Biological markers performable in routine practice and able to predict the clinical outcome of advanced non-small cell lung cancer （NSCLC） treated with gefitinib are urgently needed. Methods： We analyzed EGFR / HER2 / HER3 primary tumour immunohistochemical expression in a prospective and consecutive series of 90 Chinese patients. Platinumpretreated patients received a 250 mg oral dose of gefitinib once daily until disease progression; EGFR / HER2 / HER3 tumour status was related with the clinical outcome in terms of response rate （RR）, time to disease progression （TTP）, and overall survival （OS）. Results： A high expression （scores 2-3） of EGFR, HER2 and HER3 was venfied in 16.7%, 43.3% and 21.1% of tumors, respectively. EGFR and HER3 status were not significantly related with response, while the HER2 overexpression result was significantly associated with a higher RR （35.9% vs. 15.7%, P = 0.027）. The RR in the 13 patients with both HER2 and HER3 expression was also significantly higher than in the other 77 patients （53.8% vs. 22.1%, P = 0.036）. EGFR / HER2 / HER3 status was not significantly correlated with TTP or OS. Conclusion： The HER2 immunohistochemical expression can play a role in the clinical management of Chinese patients with advanced NSCLC who are candidates for gefitinib therapy.

The preparation of anti-hnRNP A2/B1 polyclonal antibody and its potential application in non-small cell lung cancer

Objective： In order to evaluate potential application for diagnosis and prognosis of non-small cell-lung cancer （NSCLC）, as well as to determine its role in the pathogenesis of the disease, we prepared anti-human hnRNPA2/B1 potyclonal antibody. Methods： Prokaryotic expression vector of pET28a （＋）-hnRNP A2/B1 was constructed and bansformed into E.coli BL21. The recombinant protein induced by IPTG was purified and injected to rabbits for antibody preparation. Expression of hnRN P A2/B1 was examined in 45 tissues of NSCLC and 16 inflammatory pseudotumor tissues of lung by immunohistochemistry with the antibody. The commercial hnRNP A2/B1 monoclonal antibody was used as a controI.Results： （1） Polyclonal an-tibody against hnRNP A2/B1 with high title was obtained. （2） The positive staining in NSCLC tissues was 62.22%, which was substantially higher than that in normal tissues （40%, P = 0.035） or inflammatory pseudotumor tissues （31.25%, P=0.033）. （3） Expression of hnRNP A2/B1 positively correlated with age and the history of smoking, whereas it negatively correlated with differentiation staging of tumors. （4） Follow-up study showed that the survival time of patients with positive staining was significantly shorter than that of patients without hnRNP A2/B1 expression （P=0.048）. Conclusion： It is successful to make the recombinant protein and prepare the polyclonal antibody agonist human hnRNP A2/B1. It may be a valuable marker for the diagnosis and prognosis of NSCLC. Our results provide a basis for further study in clinical application.

The expressions and clinical significance of P53, C-erbB-2 and VEGF in non-small cell lung cancer

Objective： To investigate the expressions and the clinical significance of P53, C-erbB-2 and vascular endothelial growth factor （VEGF） in non-small cell lung cancer （NSCLC）. Methods： 121 specimens of NSCLC were examined for P53, C-erbB-2 and VEGF by immunohistochemical staining. Results： The positive rates of P53, C-erbB-2 and VEGF in the carcinomatous tissue were 43%, 39% and 31% respectively. P53 gene protein expression in lung cancer was significantly related to histological type and P-TNM staging of lung cancer patients （P 〈 0.05）, and was not associated with the sex, age, the size of primary cancer, lymph node metastasis and cell differentiation （P 〉 0.05）. C-erbB-2 gene protein expression in lung cancer was closely related to histological type and cell differentiation （P 〈 0.05）, and was not associated with the sex, age, the size of primary cancer, lymph node metastasis and P-TNM staging of lung cancer patients （P 〉 0.05）. VEGF in lung cancer was only closely related to cell differentiation （P 〈 0.05）, and was not associated with the sex, age, the size of pdmary cancer, lymph node metastasis, histological type and P-TNM staging of lung cancer patients （P 〉 0.05）. Conclusion： It is possible for P53, C-erbB-2 and VEGF to play an important role in the oncogenesis and development of non-small cell lung cancer.

Effects of Chemotherapy on Peripheral Blood NK Cell Receptor NKG2D and Related Immune Cytokines in Patients with Non-Small Cell Lung Cancer

Objective: To analyze the effect of chemotherapy on peripheral blood NK cell receptor NKG2D and related immune cytokines (IL-12, IL-15, IL-18) in patients with non-small cell lung cancer (NSCLC). Methods: A total of 48 patients with NSCLC who visited the Oncology Department of the Affiliated Hospital of Chengde Medical College from September 2018 to September 2019 were selected as the study subjects. Changes in the expression levels of NKG2D, IL-12, IL-15 and IL-18 in peripheral blood of patients at different time points (before chemotherapy, after the first chemotherapy and after the second chemotherapy) were analyzed to investigate the correlation between NKG2D and IL-12, IL-15 and IL-18 in peripheral blood at each time point. Results: The expression levels of NKG2D, IL-15, and IL-18 in the peripheral blood of the patient before chemotherapy, after the first chemotherapy, and after the second chemotherapy gradually decreased. After the first chemotherapy and the second chemotherapy, the peripheral blood IL-12 was significantly lower than before chemotherapy, and IL-12 in peripheral blood after the second chemotherapy was slightly increased compared with that after the first chemotherapy. The comparison of each factor at different time points was statistically significant (all P<span style="font-family: ">0.05). Pearson correlation analysis showed that after the first chemotherapy, NKG2D in peripheral blood was positively correlated with IL-18 (r = 0.342, P = 0.031);after the second chemotherapy, NKG2D in peripheral blood was positively correlated with IL-18 (r = 0.411, P = 0.023), negatively correlated with IL-15 (r = -0.451, P = 0.001). Conclusion: There was no significant change in the number of NK cells in the peripheral blood of NSCLC patients after chemotherapy, while NKG2D and related immune cytokines decreased, which may be one of the mechanisms for the suppression of immune function in patients, and this provides a potential target for immunotherapy in patients.

FOXM1 and UBE2C Are Distinct Biomarkers for Non-Small Cell Lung Cancer Survival Prediction: Data-Mining Based on ONCOMINE

Non-small cell lung cancer (NSCLC) remains to be primary reason of tumor deaths in the past few decades. The mortality of this malignancy could be reduced by developing new prognostic biomarkers and discovering novel therapeutic biological target. Here, we studied the mRNA expression of FOX gene family and UBE2C in different types of cancer compared with normal tissue through ONCOMINE differential analysis. CCLE analysis was mined to explore the expression profiles of target genes in different tumor cells. GEPIA was used to discover the expression of target genes in different subtypes and the correlations with lung cancer stage. The prognostic values of FOXM1 and UBE2C were further investigated through Kaplan-Meier plotter analysis. It showed that FOXA1, FOXD1 and FOXM1 were dramatically high expressed in NSCLC comparing with normal lung tissues. Besides, the expression of FOXM1 was significantly associated with UBE2C. Furthermore, the overexpression of FOXM1 and UBE2C were correlated to shorter survival in lung adenocarcinoma (LAC) instead of lung squamous cell carcinoma (LSCC). Hence, we could draw a conclusion that FOXM1 and UBE2C are distinguished biomarkers and crucial prognostic indicators for lung adenocarcinoma patients.

Pyrotinib in HER2 heterogeneously mutated or amplified advanced non-small cell lung cancer patients:a retrospective real-world study(PEARL)

Human epidermal growth factor receptor 2(HER2)amplification or activating mutations are found in 1.6%–4%of non-small cell lung cancer(NSCLC).Pyrotinib has been reported to have better potency in NSCLC patients with HER2 exon 20 insertion(ex20ins)mutations;however,more clinical evidence is urgently needed to guide pyrotinib-based therapy in NSCLC with HER2 amplification or heterogeneous mutations.We retrospectively analyzed advanced NSCLC patients with HER2 amplification or mutations who were treated with pyrotinib-based therapy between September 25,2018 and October 30,2020 in our hospital.Molecular dynamics simulation was used to explore the bioactive conformation and binding mechanisms of pan-ErbB tyrosine kinase inhibitors(TKIs)including pyrotinib for different HER2 ex20ins variants.In this study,79 eligible patients were included with 70 ex20ins variants,6 missense mutations and 3 primary HER2 amplifications identified.A775_G776insYVMA insertion was the most common observed subtype.The median progression-free survival(mPFS)was 5.8(95%CI:4.1–7.4)months.Use of pyrotinib-based therapy in first-/second-line settings showed a significantly better prognosis than that observed in third-line settings or above(mPFS:9.1 vs.4.4 months;P=0.0003).Compared with HER2 amplification and exon 20 non-YVMA insertion variants,patients with HER2 missense mutations had a visible mPFS benefit(12.2 vs.6.8 vs.5.2 months).Computational docking simulations revealed that pyrotinib failed to interact with the specific insertion variant P780_Y781insGSP.These results indicated that pyrotinib-based therapy exhibited good anti-tumor activity and acceptable safety profile in HER2-altered advanced NSCLC.

Ent-11α-Hydroxy-15-oxo-kaur-16-en-19-oic-acid Inhibits Growth of Human Lung Cancer A549 Cells by Arresting Cell Cycle and Triggering Apoptosis

Objective： To examine the apoptotic effect of ent-llα-hydroxy-15-oxo-kaur-16-en-19-oic-acid （5F）, a compound isolated from Pteris semipinnata L （PsL）, in human lung cancer A549 cells. Methods： A549 cells were treated with 5F （0-80 lag/ml） for different time periods. Cytotoxicity was examined using a Ml-I- method. Cell cycle was examined using propidium iodide staining. Apoptosis was examined using Hoechst 33258 staining, enzyme-linked immunosorbent assay （ELISA） and caspase-3 activity analysis. Expression of representative apoptosis-related proteins was evaluated by Western blot analysis. Reactive oxygen species （ROS） level was measured using standard protocols. Potential interaction of 5F with cisplatin was also examined. Results： 5F inhibited the proliferation of A549 cells in a concentration- and time-dependent manner. 5F increased the accumulation of cells in sub-G1 phase and arrested the cells in the G2 phase. Exposure to 5F induced morphological changes and DNA fragmentation that are characteristic of apoptosis. The expression of p21 was increased. 5F exposure also increased Bax expression, release of cytochrome c and apoptosis inducing factor （AIF）, and activation of caspase-3. 5F significantly sensitized the cells to cisplatin toxicity. Interestingly, treatment with 5F did not increase ROS, but reduced ROS production induced by cisplatin. Conclusion： 5F could inhibit the proliferation of A549 cells by arresting the cells in G2 phase and by inducing mitochondrial-mediated apoptosis.

Flotillin1 promotes EMT of human small cell lung cancer via TGF-β signaling pathway

Objective:Small cell lung carcinoma(SCLC)is considered one of the most aggressive types of lung cancer due to its rapid growth and early metastasis.No tumor markers or therapeutic targets have been demonstrated to be specific or effective in SCLC to date.This study aims to evaluate the potential of Flotillin1(Flot1)as a target of SCLC treatment.Methods:Flot1 expression level in the tissue of SCLC and other tissue of lung disease was detected using immunohistochemical staining.Transwell and Matrigel assays were employed to examine migration and invasion of cancer cells.Flow cytometry and xCELLigence system were used to evaluate cell apoptosis and cell viability,respectively.Expression levels of Flot1,epithelialmesenchymal transition(EMT)marker E-cadherin,vimentin,cyclinD1,TGF-β-Smad2/3,and p-AKT were examined using Western blot.Furthermore,xenograft tumor in nude mice was used to evaluate the growth and metastasis of NCI-H446 cells in vivo.Results:Our results demonstrated that Flot1 is highly expressed in SCLC samples and that its expression correlates strongly with clinical stage,distant metastasis,and poor survival.The knockdown of Flot1 decreased the growth,migration,and invasiveness of SCLC cells and reversed EMT phenotype in vitro and in vivo,while enhanced Flot1 expression exhibited the opposite behavior.Gene expression profile analysis demonstrated that Flot1-regulated genes frequently mapped to the AKT and TGF-β-Smad2/3pathways.Our results further revealed that Flot1 affected the progression of SCLC via regulation of EMT progression.Conclusions:These findings indicated an oncogenic role of Flot1 via promoting EMT in SCLC and suggested its potential as a tumor marker and prognostic indicator.