Yu Deng

miR-138-5p reverses gefitinib resistance in non-small cell lung cancer cells via negatively regulating G protein-coupled receptor 124.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) such as gefitinib are clinically effective treatments for non-small cell lung cancer (NSCLC) patients with EGFR activating mutations. However, therapeutic effect is ultimately limited by the development of acquired TKI resistance. MicroRNAs (miRNAs) represent a category of small non-coding RNAs commonly deregulated in human malignancies. The aim of this study was to investigate the role of miRNAs in gefitinib resistance. We established a gefitinib-resistant cell model (PC9GR) by continually exposing PC9 NSCLC cells to gefitinib for 6 months. MiRNA microarray screening revealed miR-138-5p showed the greatest downregulation in PC9GR cells. Re-expression of miR-138-5p was sufficient to sensitize PC9GR cells and another gefitinib-resistant NSCLC cell line, H1975, to gefitinib. Bioinformatics analysis and luciferase reporter assay showed that G protein-coupled receptor124 (GPR124) was a direct target of miR-138-5p. Experimental validation demonstrated that expression of GPR124 was suppressed by miR-138-5p on protein and mRNA levels in NSCLC cells. Furthermore, we observed an inverse correlation between the expression of miR-138-5p and GPR124 in lung adenocarcinoma specimens. Knockdown of GPR124 mimicked the effects of miR-138-5p on the sensitivity to gefitinib. Collectively, our results suggest that downregulation of miR-138-5p contributes to gefitinib resistance and that restoration of miR-138-5p or inhibition GPR124 might serve as potential therapeutic approach for overcoming NSCLC gefitinib resistance.

Estrogen and insulin-like growth factor 1 synergistically promote the development of lung adenocarcinoma in mice

Estrogen receptor (ER) and insulin‐like growth factor‐1 receptor (IGF‐1R) signaling are implicated in lung cancer progression. Based on their previous findings, the authors sought to investigate whether estrogen and IGF‐1 act synergistically to promote lung adenocarcinoma (LADE) development in mice. LADE was induced with urethane in ovariectomized Kunming mice. Tumor‐bearing mice were divided into seven groups: 17β‐estradiol (E2), E2+fulvestrant (Ful; estrogen inhibitor), IGF‐1, IGF‐1+AG1024 (IGF‐1 inhibitor), E2+IGF‐1, E2+IGF‐1+Ful+AG1024 and control groups. After 14 weeks, the mice were sacrificed, and then the tumor growth was determined. The expression of ERα/ERβ, IGF‐1, IGF‐1R and Ki67 was examined using tissue‐microarray‐immunohistochemistry, and IGF‐1, p‐ERβ, p‐IGF‐1R, p‐MAPK and p‐AKT levels were determined based on Western blot analysis. Fluorescence‐quantitative polymerase chain reaction was used to detect the mRNA expression of ERβ, ERβ2 and IGF‐1R. Tumors were found in 93.88% (46/49) of urethane‐treated mice, and pathologically proven LADE was noted in 75.51% (37/49). In the E2+IGF‐1 group, tumor growth was significantly higher than in the E2 group (p < 0.05), the IGF‐1 group (p < 0.05) and control group (p < 0.05). Similarly, the expression of ERβ, p‐ERβ, ERβ2, IGF‐1, IGF‐1R, p‐IGF‐1R, p‐MAPK, p‐AKT and Ki67 at the protein and/or mRNA levels was markedly higher in the ligand group than in the ligand + inhibitor groups (all p < 0.05). This study demonstrated for the first time that estrogen and IGF‐1 act to synergistically promote the development of LADE in mice, and this may be related to the activation of the MAPK and AKT signaling pathways in which ERβ1, ERβ2 and IGF‐1R play important roles.

MiR-509-5p suppresses the proliferation, migration, and invasion of non-small cell lung cancer by targeting YWHAG

Dysregulation of microRNAs (miRNAs) expression is prevalent in human malignancy development and progression. As previously reported, miR-509-5p has been identified as a tumor suppressor in various cancers, but its role and functional mechanism in non-small lung cancer (NSCLC) remains unknown. In the present study, we found that miR-509-5p was significantly downregulated in NSCLC tissues and cell lines using Quantitative real-time PCR (qRT-PCR). In addition, overexpression of miR-509-5p markedly inhibited the proliferation, migration, invasion and phosphorylation of Akt of NSCLC cells. Moreover, we identified tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma (YWHAG) as a direct target of miR-509-5p. Knockdown of YWHAG mimicked the inhibitory effects of miR-509-5p on NSCLC cells for proliferation and motility. Finally, the negative correlation between miR-509-5p and YWHAG expression in NSCLC specimens was demonstrated. Taken together, our present study revealed the tumor suppressive role of miR-509-5p in NSCLC by targeting YWHAG, suggesting that miR-509-5p/YWHAG axis might be considered as a novel and potential target for clinical diagnosis and therapeutics of NSCLC.

USP2a Supports Metastasis by Tuning TGF-β Signaling

TGF-β has been demonstrated to promote tumor metastasis, and the regulatory mechanisms are poorly understood. Here, we report the role of USP2a in promoting metastasis by facilitating TGF-β-triggered signaling. USP2a interacts with TGFBR1 and TGFBR2 upon TGF-β stimulation and removes K33-linked polyubiquitin chains from Lys502 of TGFBR1, promoting the recruitment of SMAD2/3. Simultaneously, TGFBR2 phosphorylates Ser207/Ser225 of USP2a, leading to the disassociation of SMAD2/3 from TGFBR1. The phosphorylation of USP2a and SMAD2 is positively correlated in human tumor biopsies, and USP2a is hyper-phosphorylated in lung adenocarcinomas with lymph node invasion. Depletion or pharmacologic inhibition of USP2a dampens TGF-β-triggered signaling and metastasis. Our findings have characterized an essential role of USP2a as a potential target for treatment of metastatic cancers.

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 miR-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 miR-124 depletion induced gefitinib resistance, and miR-124 overexpression sensitized gefitinib-resistant cells to gefitinib. Mechanistic analysis revealed that miR-124 decreased SNAI2 and STAT3 expression by directly targeting their 3UTRs and that knocking down SNAI2 or STAT3 partly reversed the gefitinib resistance induced by miR-124 depletion. Our data demonstrate that the miR-124 plays a new critical role in acquired resistance to gefitinib and that the manipulation of miR-124 might provide a therapeutic strategy for reversing acquired gefitinib resistance.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 miR-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 miR-124 depletion induced gefitinib resistance, and miR-124 overexpression sensitized gefitinib-resistant cells to gefitinib. Mechanistic analysis revealed that miR-124 decreased SNAI2 and STAT3 expression by directly targeting their 3UTRs and that knocking down SNAI2 or STAT3 partly reversed the gefitinib resistance induced by miR-124 depletion. Our data demonstrate that the miR-124 plays a new critical role in acquired resistance to gefitinib and that the manipulation of miR-124 might provide a therapeutic strategy for reversing acquired gefitinib resistance.

Immunohistochemistry with a novel mutation-specific monoclonal antibody as a screening tool for the EGFR L858R mutational status in primary lung adenocarcinoma

Epidermal growth factor receptor (EGFR) mutation status is the best predictor of patient response to treatments with tyrosine kinase inhibitors in primary lung adenocarcinoma and is typically analyzed by DNA-based techniques, such as direct DNA sequencing and allele-specific PCR. Recently, however, two mutation-specific antibodies against delE746-A750 in exon 19 and L858R in exon 21 have opened the door for a more convenient and more efficient strategy to determine EGFR mutation status. To evaluate the clinical application of a new mutation-specific mouse monoclonal antibody for EGFR (L858R), we performed immunohistochemistry (IHC) studies with tumor samples from primary lung adenocarcinoma in retrospective and validation settings. A total of 215 cases of primary lung adenocarcinoma were examined and compared using a combination of DNA-based techniques (direct DNA sequencing and/or allele-specific PCR) and protein-based IHC. IHC staining was assessed on a 0 to 3+ score scale, and a cutoff value of 2+ was used as positive by IHC. In the retrospective setting, statistical analyses of the data showed that the sensitivity of IHC was 90.9xa0% and the specificity was 96.8xa0%. Findings from the validation study demonstrated that the sensitivity and specificity of IHC were 88.2xa0% and 100xa0%, respectively. IHC with the novel mutation-specific antibody could be used as a screening method to assess the EGFR L858R mutation status in primary lung adenocarcinoma.

Association of preoperative serum IGF- I concentration with clinicopathological parameters in patients with non-small cell lung cancer

SummaryInsulin-like growth factor-I (IGF-I) is a mitogenic and anti-apoptotic factor. Serum IGF-I concentration is related to some cancer risk and tumor progression. The aim of this research was to study the association of preoperative serum IGF-I concentration with clinicopathological parameters and prognosis of non-small cell lung cancer (NSCLC). Preoperative serum IGF-I concentration was measured in 80 consecutive patients with NSCLC who underwent radical lung cancer resection, and 45 patients with benign pulmonary lesion (BPL) by using enzyme linked immunosorbent assay (ELISA). The results showed that the serum IGF-I concentration was elevated and correlated with clinicopathological parameters and overall survival (OS) in NSCLC patients. Serum IGF-I concentration was significantly higher in patients with NSCLC than in those with BPL. The IGF-I concentrations were significantly higher in NSCLC patients with ≥T2, N1-3, and in IIIA-IV but not in those with <T2, N0, or IA-IIB. The increased serum IGF-I concentration was significantly correlated with poor prognosis. Our data show the positive correlation between IGF-I serum concentration and the tumor size for the first time. It seems that IGF-I related to the progression of lung cancer may depend on autocrine/paracrine function. In addition, our study reveals that higher serum IGF-I concentration is correlated with larger tumor size, advanced stages, local lymph node metastasis and worse prognosis, indicating that endocrine IGF-I is also important for the progression for NSCLC.Insulin-like growth factor-I (IGF-I) is a mitogenic and anti-apoptotic factor. Serum IGF-I concentration is related to some cancer risk and tumor progression. The aim of this research was to study the association of preoperative serum IGF-I concentration with clinicopathological parameters and prognosis of non-small cell lung cancer (NSCLC). Preoperative serum IGF-I concentration was measured in 80 consecutive patients with NSCLC who underwent radical lung cancer resection, and 45 patients with benign pulmonary lesion (BPL) by using enzyme linked immunosorbent assay (ELISA). The results showed that the serum IGF-I concentration was elevated and correlated with clinicopathological parameters and overall survival (OS) in NSCLC patients. Serum IGF-I concentration was significantly higher in patients with NSCLC than in those with BPL. The IGF-I concentrations were significantly higher in NSCLC patients with ≥T2, N1-3, and in IIIA-IV but not in those with <T2, N0, or IA-IIB. The increased serum IGF-I concentration was significantly correlated with poor prognosis. Our data show the positive correlation between IGF-I serum concentration and the tumor size for the first time. It seems that IGF-I related to the progression of lung cancer may depend on autocrine/paracrine function. In addition, our study reveals that higher serum IGF-I concentration is correlated with larger tumor size, advanced stages, local lymph node metastasis and worse prognosis, indicating that endocrine IGF-I is also important for the progression for NSCLC.

Expression and clinical significance of insulin-like growth factor 1 in lung cancer tissues and perioperative circulation from patients with non-small-cell lung cancer

OBJECTIVEnWe explored the role of insulin-like growth factor 1 (igf-1) in the development of lung cancer.nnnMETHODSnWe used immunohistochemistry to measure the expression of igf-1 and igf-1 receptor (igf-1r) in specimens of tissue and perioperative circulation from 80 patients with primary non-small-cell lung cancer (nsclc) and from 45 patients with benign pulmonary lesions (bpls). Correlations of those measurements with clinicopathologic characteristics and clinical follow-up were analyzed. Circulating igf-1 was measured before and after surgery in all patients.nnnRESULTSnCompared with bpl specimens, nsclc specimens showed overexpression of igf-1and igf-1r (p < 0.001). The expression levels of igf-1 and igf-1r were significantly associated with advanced-stage disease (p = 0.034 and 0.029 respectively) and lymph node metastasis (p = 0.012 and 0.017 respectively), and expression of igf-1 correlated with tumour differentiation and tumour diameter (p = 0.011 and 0.021 respectively). Specimens positive for igf-1 or igf-1r were significantly correlated with shorter patient survival (p = 0.0012 and 0.0016 respectively). After surgery, circulating igf-1 was significantly elevated in patients with bpl (p = 0.0346) and significantly lower in patients with nsclc (p = 0.0030), especially in those with advanced-stage disease, a larger tumour size, regional lymphoid node metastasis, or lesser differentiation (p = 0.0092, 0.0051, 0.0131, and p < 0.001 respectively).nnnCONCLUSIONSnIn nsclc, igf-1 and igf-1r are upregulated, and expression of those factors is correlated with tumour progression and prognosis in nsclc patients. Radical resection of nsclc can directly influence the serum concentration of igf-1. Autocrine/paracrine igf-1 might be playing an important role in the development of lung cancer.

Knockdown of lncRNA-XIST enhances the chemosensitivity of NSCLC cells via suppression of autophagy

Drug resistance is the major factor contributing to the failure of chemotherapy in non-small cell lung cancer (NSCLC) patients. Emerging evidence suggests that autophagy plays a vital role in the chemoresistance of many types of tumors. However, the exact mechanism underlying the chemoresistance of NSCLC is still elusive, and it is unclear whether lncRNA-XIST is involved in autophagy and chemoresistance of NSCLC. In the present study, we demonstrated that lncRNA-XIST was overexpressed in NSCLC tumor samples, and knockdown of lncRNA-XIST significantly decreased autophagy by regulation of ATG7 as determined by qPCR and by western blotting. Furthermore, we found that miR-17 was upregulated following knockdown of lncRNA-XIST, and miR-17 mimics decreased the protein levels of ATG7 by directly targeting the 3-untranslated region of ATG7 mRNA as determined by RT-qPCR and by western blotting. Furthermore, we found that the expression level of lncRNA-XIST was markedly increased in cisplatin-resistant A549 cells as determined by q-PCR. Knockdown of lncRNA-XIST restored the chemosensitivity of cisplatin-resistant A549 cells to cisplatin, which was reversed by miR-17 inhibitor and overexpression of ATG7 as determined by CCK8 assays. This study provides evidence that lncRNA-XIST may be a potential marker of poor response to cisplatin chemotherapy in NSCLC patients and the pathway ‘lncRNA-XIST/miR-17/autophagy’ may be a promising target for patients with chemoresistant NSCLC.

Use of lung-preserving surgery in left inflammatory bronchial occlusion and distal atelectasis: preliminary results

OBJECTIVESnLung-preserving surgery was proved to be effective and safe to treat patients with benign bronchial strictures. However, this surgical treatment has been rarely reported in patients with complete occlusion in the left main bronchus. The aim of this study was to assess the value of this procedure and report our experience in the treatment of these patients with left atelectasis caused by inflammatory bronchial occlusion.nnnMETHODSnWe reviewed and analysed the medical records of 8 patients who had undergone left main bronchus sleeve resection for symptomatic left atelectasis caused by inflammatory bronchial occlusion from May 2007 to April 2011.nnnRESULTSnEight patients (3 men and 5 women) with a medical history of active pulmonary tuberculosis were involved in this study. The median age was 23 years. Parenchyma-sparing left main bronchus resection was performed in 4 patients, 1 of whom received partial wedge resection in the lingual lobe. Left main bronchus sleeve resection plus superior lobectomy was performed in 2 patients and left main bronchus sleeve resection plus left inferior lobectomy in 2 patients, 1 of whom received additional partial wedge resection of the lingual lobe. The procedure was completed successfully in all 8 patients without postoperative deaths. The mean follow-up time was 49.3 months, ranging from 23 to 69 months. No major complications, including stenosis and atelectasis, were observed during the follow-up period. The symptoms of pulmonary atelectasis disappeared and pulmonary ventilation function improved significantly.nnnCONCLUSIONSnIn symptomatic patients with left atelectasis caused by inflammatory bronchial occlusion, lung-preserving surgery is an effective and safe surgical treatment.