Yasushi Shintani

Overexpression of ADAM9 in Non-Small Cell Lung Cancer Correlates with Brain Metastasis

The “a disintegrin and metalloprotease” (ADAM) family contributes to regulation of the cell–cell and cell–matrix interactions that are critical determinants of malignancy. To determine the relationship between metastasis and ADAM proteins, we compared the mRNA levels of ADAM9, -10, -12, -15, and -17 in sublines of an EBC-1 lung cancer cell line that were highly metastatic to either brain or bone. ADAM9 mRNA levels were significantly higher in highly brain-metastatic sublines than in the parent or highly bone-metastatic sublines. To elucidate the role of ADAM9 in brain metastasis, we stably transfected A549 and EBC-1 cells with a full-length ADAM9 expression vector. Compared with mock-transfectants, ADAM9 overexpression resulted in increased invasive capacity in response to nerve growth factor, increased adhesion to brain tissue, and increased expression of integrin α3 and β1 subunits. Administration of the anti-β1 monoclonal antibody attenuated this increase in invasive and adhesive activity. Intravenous administration of ADAM9-overexpressing A549 cells to mice resulted in micrometastatic foci in the brain and multiple metastatic colonies in the lungs. In contrast, administration of parent and mock-transfected A549 cells to mice resulted in lung tumors without brain metastasis. These results suggest that ADAM9 overexpression enhances cell adhesion and invasion of non-small cell lung cancer cells via modulation of other adhesion molecules and changes in sensitivity to growth factors, thereby promoting metastatic capacity to the brain.

Epithelial to Mesenchymal Transition Is a Determinant of Sensitivity to Chemoradiotherapy in Non-Small Cell Lung Cancer

BACKGROUND The epithelial to mesenchymal transition (EMT) is a fundamental biological process during which epithelial cells change to a mesenchymal phenotype; it has a profound impact on cancer progression. The purpose of this study was to clarify the role of EMT in the sensitivity of non-small cell lung cancer (NSCLC) to chemoradiotherapy (CRT). METHODS We evaluated the correlation between EMT and sensitivity to chemotherapy or radiotherapy using NSCLC cells induced to undergo EMT with epidermal growth factor or transforming growth factor-β1. Immunohistochemistry was used to examine the expression of EMT markers, E-cadherin, cytokeratin, N-cadherin, and vimentin in 50 tumor specimens obtained from patients with NSCLC both before and after CRT. RESULTS The EMT resulted in increased malignant potential and reduced sensitivity to cisplatin and paclitaxel in NSCLC cells. Furthermore, chronic exposure to cisplatin, paclitaxel, or radiation altered the cells into therapy-resistant sub-lines that showed phenotypic changes such as a spindle-cell shape and increased EMT marker expression. Also, decreased expression of epithelial markers and upregulation of mesenchymal markers were detected in surgically resected specimens after CRT compared with biopsy specimens obtained before treatment. The disease-free survival rate of patients with EMT marker-positive tumors was significantly lower than that of those with EMT marker-negative tumors. CONCLUSIONS The EMT marker expression was detected in NSCLC tumors after CRT, indicating that EMT changes are associated with insensitivity to CRT. New therapeutic combinations using EMT-signaling inhibitors may be needed to circumvent the resistance of some types of cancer to CRT.

New prognostic indicator for non‐small‐cell lung cancer, quantitation of thymidylate synthase by real‐time reverse transcription polymerase chain reaction

Thymidylate synthase (TS) is an enzyme that catalyzes an important DNA biosynthesis process. The gene expression of TS has not been reported in non‐small‐cell lung cancer (NSCLC) patients. To clarify the correlation between TS mRNA levels and clinicopathological features of NSCLC, we examined 70 Stage I and II NSCLC patients for intra‐tumoral expression of TS using TaqMan reverse transcription polymerase chain reaction (RT‐PCR) assay and immunohistochemistry methods. We also investigated the TS promoter 28 bp polymorphism in 48 cancer tissues using PCR amplification of genomic DNA. Lung cancer tissue showed higher TS mRNA levels than normal lung tissue (Mann‐Whitney U‐tests; p = 0.0020). Further, TS mRNA expression was correlated with immunohistochemical TS expression (p = 0.029). We obtained 2 different DNA fragments, which indicated triple‐repeat (3R) and double‐repeat (2R) type alleles. Cancer tissues with the 3R/3R genotype showed significantly higher TS mRNA levels as compared to those with other genotypes (p = 0.0019). The TS genotype was also correlated with immunohistochemical TS expression (χ2 test; p = 0.0079). The disease‐free survival of the low TS mRNA level group was significantly better than those with high TS mRNA levels (log‐rank test; p = 0.010), however, there were no significant differences found by immunohistochemical evaluation (p = 0.34) or TS genotype analysis (p = 0.11). A multivariate analysis revealed that high TS mRNA levels independently contributed to disease‐free survival. The quantitation of TS mRNA levels is clinically more sensitive and useful for determining the prognosis of Stage I and II NSCLC patients than an immunohistochemical evaluation.

MicroRNA-mediated downregulation of mTOR/FGFR3 controls tumor growth induced by Src-related oncogenic pathways.

The tyrosine kinase c-Src is upregulated in various human cancers, but the molecular mechanisms underlying c-Src-mediated tumor growth remain unclear. Here we examined the involvement of microRNAs in the c-Src-mediated tumor growth. Microarray profiling revealed that c-Src activation downregulates a limited set of microRNAs, including miR-99a, which targets oncogenic mammalian target of rapamycin (mTOR) and fibroblast growth factor receptor 3 (FGFR3). Re-expression of miR-99a suppressed tumor growth of c-Src-transformed cells, and this effect was restored by the overexpression of mTOR. The downregulation of miR-99a was also observed in epidermal growth factor- and Ras-transformed cells, and it was suppressed by inhibiting the mitogen-activated protein kinase (MAPK) pathway. Furthermore, miR-99a downregulation is associated with mTOR/FGFR3 upregulation in various human lung cancer cells/tissues. The tumorigenicity of these cells was suppressed by the introduction of miR-99a. These findings suggest that the miR-99a-mTOR/FGFR3 pathway is crucial for controlling tumor growth in a wide range of human cancers that harbor upregulation of the Src-related oncogenic pathways.

Predictive factors for postoperative acute exacerbation of interstitial pneumonia combined with lung cancer

PurposePostoperative acute exacerbation (AE) of usual interstitial pneumonia (UIP) is a serious complication in the surgical treatment for primary lung cancer combined with UIP. The purpose of this study was to determine the predictors of AE of UIP after a major lung resection.MethodsWe retrospectively collected data for 40 patients who had been operated on for lung cancer and were diagnosed as UIP based on postoperative histopathological diagnosis. We then evaluated some predictive factors related to the AE of UIP.ResultsThe incidence of postoperative AE of UIP was 15% (6/40 patients). No correlation between patients who developed AE of UIP and those who did not, in terms of preoperative C-reactive protein, white blood cell count, percentage lymphocytes, forced expiratory volume in 1 s, percentage total lung capacity, percentage diffusing capacity of lung for carbon monoxide, and the alveolar partial pressures of oxygen and carbon dioxide. Preoperative serum lactate dehydrogenase (LDH) and serum KL-6 were significantly higher and the percent vital capacity (%VC) was significantly lower in patients who developed AE of UIP than in those who did not. Furthermore, recursive descent partition analysis revealed that %VC (<80.6%) and LDH (≥241 IU/l) could distinguish patients with AE from those without AE.ConclusionPreoperative %VC plus serum LDH values were considered the predictive factors for AE of UIP after surgery for lung cancer.

Atrial natriuretic peptide prevents cancer metastasis through vascular endothelial cells

Significance Postoperative cancer recurrence is a major problem following curative cancer surgery. Perioperative systemic inflammation induces the adhesion of circulating tumor cells released from the primary tumor to the vascular endothelium of distant organs, which is the first step in hematogenous metastasis. We have previously reported that administration of atrial natriuretic peptide (ANP) during the perioperative period reduces inflammatory response and has a prophylactic effect on postoperative cardiopulmonary complications in lung cancer surgery. Here, we demonstrate that cancer recurrence after lung cancer surgery was significantly lower in ANP-treated patients than in control patients (surgery alone). We show that ANP prevents cancer metastasis by suppressing the inflammatory reaction of endothelial cells, thereby inhibiting cancer cell adhesion to vascular endothelial cells. Most patients suffering from cancer die of metastatic disease. Surgical removal of solid tumors is performed as an initial attempt to cure patients; however, surgery is often accompanied with trauma, which can promote early recurrence by provoking detachment of tumor cells into the blood stream or inducing systemic inflammation or both. We have previously reported that administration of atrial natriuretic peptide (ANP) during the perioperative period reduces inflammatory response and has a prophylactic effect on postoperative cardiopulmonary complications in lung cancer surgery. Here we demonstrate that cancer recurrence after curative surgery was significantly lower in ANP-treated patients than in control patients (surgery alone). ANP is known to bind specifically to NPR1 [also called guanylyl cyclase-A (GC-A) receptor]. In mouse models, we found that metastasis of GC-A–nonexpressing tumor cells (i.e., B16 mouse melanoma cells) to the lung was increased in vascular endothelium-specific GC-A knockout mice and decreased in vascular endothelium-specific GC-A transgenic mice compared with control mice. We examined the effect of ANP on tumor metastasis in mice treated with lipopolysaccharide, which mimics systemic inflammation induced by surgical stress. ANP inhibited the adhesion of cancer cells to pulmonary arterial and micro-vascular endothelial cells by suppressing the E-selectin expression that is promoted by inflammation. These results suggest that ANP prevents cancer metastasis by inhibiting the adhesion of tumor cells to inflamed endothelial cells.

IL-6 Amplifier, NF-κB–Triggered Positive Feedback for IL-6 Signaling, in Grafts Is Involved in Allogeneic Rejection Responses

The IL-6-amplifier first was discovered as a synergistic activation mechanism for NF-κB/STAT3 in type 1 collagen+ cells. This process is marked by the hyperinduction of chemokines and subsequent local inflammation that leads to autoimmune diseases. In this study, we show that IL-6 amplifier activation in grafts plays important roles in allogeneic graft rejection by using a tracheal heterotopic transplantation model that includes bronchiolitis obliterans, a pathological marker for chronic rejection. IL-6, epidermal growth factor, and IFN-γ all stimulate IL-6 amplifier activation, whereas CCL2, a chemotactic factor for Th1 cells, was one of the amplifier’s main targets. Interestingly, IFN-γ hyperinduced CCL2 in type 1 collagen+ cells via the IL-6 amplifier at least in vitro. In addition, we detected IL-6, CCL2, phospho-STAT3, and phospho-NF-κB in epithelial type 1 collagen+ cells of allogeneic tracheal grafts. These results show that IL-6 amplifier activation in grafts plays a critical role for graft rejection responses after allogeneic transplantation, including chronic rejection. From these results, we consider whether the IL-6 amplifier in grafts might be a valuable therapeutic target for the prevention of transplant rejection, including chronic rejection.

Tetraspanin CD151 Protects against Pulmonary Fibrosis by Maintaining Epithelial Integrity

RATIONALE Idiopathic pulmonary fibrosis (IPF) is a chronic pulmonary disorder of unknown etiology with few treatment options. Although tetraspanins are involved in various diseases, their roles in fibrosis have not been determined. OBJECTIVES To investigate the role of tetraspanin CD151 in pulmonary fibrosis. METHODS CD151 knockout (KO) mice were studied by histological, biochemical, and physiological analyses and compared with wild-type mice and CD9 KO mice. Further mechanistic analyses were performed in vitro, in vivo, and on samples from patients with IPF. MEASUREMENTS AND MAIN RESULTS A microarray study identified an enrichment of genes involved in connective tissue disorders in the lungs of CD151 KO mice, but not in CD9 KO mice. Consistent with this, CD151 KO mice spontaneously exhibited age-related pulmonary fibrosis. Deletion of CD151 did not affect pulmonary fibroblast functions but instead degraded epithelial integrity via attenuated adhesion strength on the basement membrane; CD151-deleted alveolar epithelial cells exhibited increased α-SMA expression with activation of p-Smad2, leading to fibrotic changes in the lungs. This loss of epithelial integrity in CD151 KO lungs was further exacerbated by intratracheal bleomycin exposure, resulting in severe fibrosis with increased mortality. We also observed decreased numbers of CD151-positive alveolar epithelial cells in patients with IPF. CONCLUSIONS CD151 is essential for normal function of alveolar epithelial cells; loss of CD151 causes pulmonary fibrosis as a result of epithelial disintegrity. Given that CD151 may protect against fibrosis, this protein represents a novel target for the treatment of fibrotic diseases.

The oncological feasibility and limitations of video-assisted thoracoscopic thymectomy for early-stage thymomas

OBJECTIVES Although video-assisted thoracoscopic thymectomy (VATS-Tx) for thymoma has been introduced, its oncological outcome remains unclear. Our institutional experience with early-stage thymoma was retrospectively reviewed to evaluate the oncological feasibility of thoracoscopic thymectomy. METHODS A retrospective review consisting of 74 patients with Masaoka Stage I and II thymomas who had undergone thymectomy was performed. Forty-five patients underwent thoracoscopic thymectomy, while 29 underwent thymectomy through the open sternotomy approach. The clinical factors associated with the surgical outcome, including tumour recurrence, were investigated. RESULTS Neither operative death nor major postoperative complications were observed. The median intraoperative blood loss and operative time of thoracoscopic thymectomy were 50 ml and 180 min, respectively. Among the patients with thymomas >5 cm, the number of patients with operative time >4 h was 9 of 26 (34.6%) in the thoracoscopic thymectomy and 1 of 21 (4.8%) in the open sternotomy groups. Pleural recurrence was observed in 3 (6.7%) patients with thymoma >5 cm only in the thoracoscopic thymectomy group. Tumour capsule injury by manipulation during the operation was recorded in 2 of these 3 patients. In 2 of the 3 cases who had tumours with cystic portions on computed tomography, a tumour capsule injury occurred due to manipulation during thoracoscopic thymectomy. CONCLUSIONS VATS-Tx for early-stage thymomas is feasible, while the indications should be carefully considered in patients with large or cystic tumours. The conventional open sternotomy approach could be recommended in patients with thymomas >5 cm to avoid capsule injury.

Risk of pleural recurrence after computed tomographic-guided percutaneous needle biopsy in stage I lung cancer patients.

BACKGROUND A computed tomographic-guided percutaneous needle biopsy (CTGNB) is useful as an option for pathologic diagnosis of lung cancer, especially in patients with peripheral small-sized nodules. We aimed to assess the risk of pleural seeding of cancer cells, leading to postoperative relapse with dissemination caused by the procedure. METHODS We investigated the clinical outcomes of 447 stage I lung cancer patients. Survival analysis was performed using the Kaplan-Meier method and a log-rank test. Pleural recurrence rates were also determined. Furthermore, propensity score matching analysis was used to reduce background bias from patient characteristics. RESULTS The 5-year, disease-free survival rate was 89.1% in patients diagnosed with CTGNB, and 85.5% in those diagnosed using a transbronchial biopsy or open lung biopsy procedure. Local recurrence with pleural dissemination was found in 8 of 13 recurrence cases (61.5%) in the CTGNB group, which was higher as compared with the transbronchial biopsy or open lung biopsy group (p < 0.01). Subset analyses of p stage IB cases and those with subpleural lesions showed that local recurrence with dissemination was significantly more frequent in the CTGNB group (p = 0.02 and p < 0.01, respectively). In patients with subpleural lesions diagnosed with CTGNB, the rate of local recurrence with dissemination was 15.4%. Propensity score matching analysis confirmed the significantly increased frequency of pleural dissemination after CTGNB. CONCLUSIONS The CTGNB procedure might increase the risk of pleural implantation in stage I lung cancer patients, especially p stage IB cases with subpleural lesions, whereas the overall disease-free survival rate was not affected by this small population of patients with recurrence.