Shigeto Nishikawa

Hypoxia-inducible factor 1 promotes chemoresistance of lung cancer by inducing carbonic anhydrase IX expression

Lung cancer treatment is difficult owing to chemoresistance. Hypoxia‐inducible factor 1 (HIF‐1) and HIF‐1‐induced glycolysis are correlated with chemoresistance; however, this is not evident in lung cancer. We investigated the effect of HIF‐1α and carbonic anhydrase IX (CAIX), a transmembrane protein neutralizing intracellular acidosis, on chemoresistance and prognosis of lung cancer patients after induction chemoradiotherapy. Associations of HIF‐1α, glucose transporter 1 (GLUT1), and CAIX with chemoresistance of lung cancer were investigated using A549 lung cancer cells under normoxia or hypoxia in vitro. HIF‐1α‐induced reprogramming of glucose metabolic pathway in A549 cells and the effects of HIF‐1 and CAIX on the cytotoxicity of vinorelbine were investigated. Immunohistochemical analyses were performed to determine HIF‐1α, GLUT1, and CAIX expression levels in cancer specimens from lung cancer patients after induction chemoradiotherapy. Hypoxia induced HIF‐1α expression in A549 cells. Moreover, hypoxia induced GLUT1 and CAIX expression in A549 cells in a HIF‐1‐dependent manner. Glucose metabolic pathway was shifted from oxidative phosphorylation to glycolysis by inducing HIF‐1α in A549 cells. HIF‐1 and CAIX induced chemoresistance under hypoxia, and their inhibition restored the chemosensitivity of A549 cells. The expression levels of HIF‐1α, GLUT1, and CAIX were associated with poor overall survival of lung cancer patients after induction chemoradiotherapy. HIF‐1 and CAIX affected the chemosensitivity of A549 cells and prognosis of lung cancer patients. Therefore, inhibition of HIF‐1 and CAIX might improve prognosis of lung cancer patients after induction chemoradiotherapy. Further analysis might be helpful in developing therapies for lung cancer.

Early and late recurrence after intentional limited resection for cT1aN0M0, non-small cell lung cancer: from a multi-institutional, retrospective analysis in Japan

OBJECTIVES In 2015, we reported the outcomes of patients undergoing intentional limited resection (ILR) for non-small-cell lung cancer (NSCLC) from a retrospective, multi-institutional large database in Japan. Here, we analyse the clinicopathological characteristics of the patients extracted from this database with late recurrence and compare them with those with early recurrence. METHODS Of 1538 patients in the database with cT1aN0M0 NSCLC, 92 (6%) had recurrence. In this study, early recurrence was defined as recurrence within 5 years and late recurrence as recurrence beyond 5 years after surgery. We compared the clinicopathological characteristics and post-recurrence survival (PRS) between patients with early and late recurrence. RESULTS Of the 92 patients with recurrence, 21 (23%) had late recurrence. Compared with the early recurrence group, there were significantly more adenocarcinomas and local recurrences in the late recurrence group (P = 0.04 for both). The 3- and 5-year PRS rates were 53 and 24%, respectively, and the median PRS period was 38 months. There were no significant differences in the PRS curves between patients with early and late recurrence (P = 0.12). Only 3 patients (0.2%) had recurrence more than 10 years after ILR. Of the 21 late-recurrence patients, 17 (81%) had tumours with a consolidation/tumour ratio (CTR) >0.25. CONCLUSIONS Late recurrence occurred in 21 (23%) of 92 patients with recurrence after ILR for cT1aN0M0 NSCLC. Late recurrence was more likely to involve adenocarcinoma and local recurrence. It is thus considered reasonable to follow patients with a CTR >0.25 for 10 years after ILR.

Abstract 1587: GEP100-Arf6 pathway enhanced by Grb2 expression plays important roles for node-metastasis of lung cancer

BACKGROUND Invasive and metastatic activities are the most challenging hallmark of cancer in clinical settings. Our previous reports have shown that GEP100 activates Arf6 by its binding to activated ErbB family receptors, such as EGFR. They also have revealed the phosphorylation at specific tyrosines in the C-terminal EGFR is necessary for the binding to the plekstrin homology (PH) domain of GEP100, which tyrosines are well known to be Grb2-binding sites as well. Additionally, this GEP100-Arf6 signal pathway is pivotal for epithelio-mesenchymal transition (EMT) leading to invasion and metastasis in various types of tumors. Here we have examined the augmentation effect of Grb2 on the binding of GEP100 with EGFR and the Arf6 activation. Furthermore, the significance of co-expression of Grb2 and GEP100 in clinical settings was analyzed MATERIALS AND METHODS GST-tagged proteins including PH domain of GEP100 or SH2/SH3 domain of Grb2 inserted into pGEX vector were expressed in bacteria with glutathione-beads purification. They were used for the mutual binding assays. A549 cells which HA-Grb2 or HA alone vector was transfected, were starved for 18hours and stimulated with EGF. And, their lysates were applied for the immunoprecipitation assay against GEP100. Then, Arf6 activities of these cells were examined using the pulldown assay with GST-tagged GGA protein. Furthermore the in vitro invasive activities of those cells were measured by Matrigel invasion assays. The expression levels of Grb2 and GEP100 of the tumor cells by immunohistochemical staining methods were examined in resected human lung adenocarcinoma specimens. The expression data of the two molecules integrated with their clinicopathological factors and EMT status information previously published were analyzed with regard to their invasive and metastatic activities. RESULTS Our results revealed that endogenous Grb2 was immunoprecipitaed with GEP100. These two molecules were physically associated through the PH domain of GEP100 and both the SH2 and N-terminal SH3 domain of Grb2, not its C-terminal SH3 domain. Exogenously aberrant expression of Grb2 in A549 lung cancer cells enhanced the association between activated EGFR and GEP100, consequently, Arf6 activation, and in vitro invasive activity, according to the expression level of Grb2. Among 239 lung adenocarcinoma specimens on tissue microarrays, 131 (55%) and 65 (27%) cases of patients were positive for Grb2 and GEP100, respectively. Tumors with double-positive for Grb2 and GEP100 (45 cases) showed significantly more aggressive EMT status (p = 0.0116) and higher node-metastatic potential (p = 0.0082, node-positive/negative; 12/33 to 7/77) than the double-negative one (84 cases). CONCLUSION Grb2 augments the binding of GEP100 to EGFR leading to Arf6 activation, and promotes lung cancer invasion and metastasis via GEP100-Arf6 pathway. Citation Format: Toshi Menju, Shigeto Nishikawa, Koji Takahashi, Shinya Neri, Takao Nakanishi, Hiroyuki Cho, Kei Shikuma, Terumasa Sowa, Makoto Sonobe, Stephan M. Feller, Hisataka Sabe, Hiroshi Date. GEP100-Arf6 pathway enhanced by Grb2 expression plays important roles for node-metastasis of lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1587.

PO-238 Clinicopathological significance of EMT markers in thymic epithelial tumours

Introduction Thymic epithelial tumours (TETs) are the relatively rare tumours originated from thymus. TETs are histologically categorised according to the WHO classification based on the morphology of epithelial tumour cells and proportion of lymphocytic involvement. Epithelio-mesenchymal transition (EMT) has reported to play pivotal roles in tumour progression including invasion/metastasis, drug resistance, and cancer stemness. However, the precise clinicopathological associations with EMT characteristics in TETs remain to be elusive. Herein, we have examined EMT markers in TETs integrated with their clinicopathological information. Material and methods A total of 109 patients with TETs were surgically resected between 2002 and 2017 in our institution. Tissue samples were collected from the paraffine-embedded tumour blocks of these patients. The 2 mm cores in diameter of the most representative areas of the tumours were selected by the pathologist and assembled to make tissue microarrays. Immunohistochemical (IHC) stainings for E-cadherin (E) and vimentin (V) were performed. P63 and pan-cytokeratin were additionally stained to detect epithelial tumour cells. The expressions of these molecules were scored and quantified to categorise the activation level of EMT into three groups based on the combination of E and V levels: Full; E-V+, Partial; E+V+ or E-V-. Null; E+V-. For statistical analyses, chi-square tests and Cox hazard models were applied. P-value less than 0.05 was considered significant. Results and discussions The median follow-up time was 54.3 months (range, 0.3–178.7). Predominant WHO histological subtypes were as follows: A;8, AB;18, B1;30, B2;30, B3;6. C;15, others;2. Pathologic stage distributions by TNM classfication were as follows: 1;80, 2;2, 3;13, 4a;7, 4b;4. The positive rates of E-cadherin or vimentin expression in epithelial tumour cells were 54.2% and 57.9%, respectively. EMT activation was significantly exhibited in indolent predominant WHO subtype, and in the early stage of pT factor and the Masaoka classification (p=0.0005, 0.0493, and 0.0366, respectively). Multivariate analyses of overall survival time including pT, pN, pM, age, and EMT levels showed that pT factor was significantly prognostic (p=0.0218), but not for EMT. Conclusion EMT activation mechanisms in TETs were inversely correlated with their histological subtype and the primary tumour progression. Further studies are necessary to elucidate tumour progression mechanisms in TETs.

The synergistic role of ATP-dependent drug efflux pump and focal adhesion signaling pathways in vinorelbine resistance in lung cancer

The vinorelbine (VRB) plus cisplatin regimen is widely used to treat non–small cell lung cancer (NSCLC), but its cure rate is poor. Drug resistance is the primary driver of chemotherapeutic failure, and the causes of resistance remain unclear. By focusing on the focal adhesion (FA) pathway, we have highlighted a signaling pathway that promotes VRB resistance in lung cancer cells. First, we established VRB‐resistant (VR) lung cancer cells (NCI‐H1299 and A549) and examined its transcriptional changes, protein expressions, and activations. We treated VR cells by Src Family Kinase (SFK) inhibitors or gene silencing and examined cell viabilities. ATP‐binding Cassette Sub‐family B Member 1 (ABCB1) was highly expressed in VR cells. A pathway analysis and western blot analysis revealed the high expression of integrins β1 and β3 and the activation of FA pathway components, including Src family kinase (SFK) and AKT, in VR cells. SFK involvement in VRB resistance was confirmed by the recovery of VRB sensitivity in FYN knockdown A549 VR cells. Saracatinib, a dual inhibitor of SFK and ABCB1, had a synergistic effect with VRB in VR cells. In conclusion, ABCB1 is the primary cause of VRB resistance. Additionally, the FA pathway, particularly integrin, and SFK, are promising targets for VRB‐resistant lung cancer. Further studies are needed to identify clinically applicable target drugs and biomarkers that will improve disease prognoses and predict therapeutic efficacies.

Pulmonary vein thrombosis after lobectomy with vein stump closure by ligation

Objectives Thrombosis in the pulmonary vein stump after a left upper lobectomy is a rare but potentially life-threatening complication, and the pulmonary vein stump length plays an important role here. We assessed the frequency and risk factors for thrombosis in patients undergoing lobectomy with division of the superior pulmonary vein using ligation. Methods We retrospectively reviewed 425 patients with primary lung cancer who underwent lobectomy or bilobectomy in our institution from 2008 to 2016, with contrast-enhanced chest computed tomography within a year after lobectomy. The superior pulmonary vein was divided by thread ligation, while the inferior pulmonary vein was divided using a linear stapler. The pulmonary vein stump length was measured using contrast-enhanced chest computed tomography. Results Four (0.9%) of the 425 patients experienced thrombosis in the pulmonary vein stump within 6 months after lobectomy. All 4 patients had undergone a left upper lobectomy, and 4.1% of this subset developed thrombus. One patient with a thrombus in the pulmonary vein stump experienced renal and cerebral infarction after a left upper lobectomy. The left superior pulmonary vein stump was significantly longer than the other pulmonary vein stumps. Conclusions Thrombosis in the pulmonary vein stump occurred in 4.1% of patients undergoing a left upper lobectomy with pulmonary vein stump closure by thread ligation, which is a relatively low frequency. Superior pulmonary vein stump closure using thread ligation might help prevent pulmonary vein stump thrombus after a left upper lobectomy.