Nrf2 is a key transcription factor for genes coding for antioxidants, detoxification enzymes, and multiple drug resistance and it also confers resistance to anticancer drugs. Here, we hypothesized that mutant p53 could upregulate Nrf2 expression at the transcriptional level, thereby conferring cisplatin resistance in non-small cell lung cancer (NSCLC). Luciferase reporter assays and real-time PCR analysis indicated that the Nrf2 promoter activity and its mRNA levels were markedly suppressed by wild-type p53, but not by mutant p53. Chromatin immunoprecipitation (ChIP) further confirmed that wild-type p53 binds at the p53 putative binding site to block Sp1 binding to the Nrf2 promoter and consequently to suppress the Nrf2 promoter activity. The MTT assay indicated that an increase in Nrf2 expression by mutant p53 is responsible for cisplatin resistance. Among the Nrf2 downstream genes, Bcl-2 and Bcl-xL contribute more strongly to Nrf2-mediated cisplatin resistance when compared with heme oxygenase 1 (HO-1). Cox regression analysis showed that patients with high-Nrf2, high-Bcl-2, high-Bcl-xL mRNA tumors were more commonly occurred unfavorable response to cisplatin-based chemotherapy than their counterparts. The prognostic significance of Nrf2 mRNA levels on OS and RFS was also observed in patients who have received cisplatin-based chemotherapy, particularly in p53-mutant patients. Collectively, mutant p53 may confer cisplatin resistance via upregulation of Nrf2 expression, and Nrf2 mRNA level may predict chemotherapeutic response and outcomes in NSCLC.

Purpose: Interleukin-10 (IL-10) determines virus persistent infection and promotes viral-associated tumor progression via tumor immune escape. However, the role of IL-10 in tumor progression and prognosis in lung adenocarcinoma remains controversial.Experimental Design: To investigate how IL-10 is regulated by HPV E6, IL-10 promoter was constructed to understand which transcriptional factor could be responsible for its transcription. To verify which molecule could be responsible for IL-10-mediated soft agar growth and invasion capability, PCR array and mechanistic strategies were conducted. IL-10 and CIP2A mRNA levels in lung tumors from patients with lung cancer were determined by real-time reverse transcription PCR. The prognostic value of both molecules on survival was estimated by Cox regression model.Results: Mechanistic studies showed that IL-10 protein and mRNA expression was decreased in E6 knockdown TL1 cells and increased in E6-overexpressing TL4 cells.

Purpose: Fas ligand (FasL) À844T/C polymorphism (rs763110) has a demonstrated association with lung cancer risk. FasL À844CC with higher FasL expression has been suggested to contribute to tumor progression via immune escape. However, the impact of FasL À844T/C polymorphism on the clinical outcome of non-small cell lung cancer (NSCLC) remains to be identified.Experimental Design: A total of 385 adjacent normal lung tissues from patients with NSCLC were collected to determine FasL À844T/C polymorphism by PCR-based restriction fragment length polymorphism. FasL mRNA and protein expression in lung tumors were evaluated by real-time PCR and immunohistochemistry. The prognostic value of FasL À844T/C polymorphism on survival and relapse was determined by Kaplan-Meier analysis and Cox proportional hazards models.Results: The FasL À844CC genotype had higher prevalence in those with advanced tumors than in those with early tumors (P ¼ 0.008). In addition, patients with the FasL À844CC genotype were more prone to tumor relapse than those with the FasL À844TTþTC genotype (62.1% vs. 37.9%, P ¼ 0.001). Multivariate Cox regression analysis showed that patients with the FasL À844CC genotype had poorer survival in terms of overall survival (OS) and relapse-free survival (RFS) than those with the FasL À844TTþTC genotype (24

PURPOSE: Foxhead box M1 (FOXM1) expression has been shown to be linked with human papillomavirus (HPV) 16/18–infected cervical cancer. However, the mechanism underlying the induction of FOXM1 in HPV 16/18–infected cancers remains elusive. EXPERIMENTAL DESIGN: The mechanistic actions of FOXM1 induced by the E6/NKX2-1 axis in tumor aggressiveness were elucidated in cellular and animal models. The prognostic value of FOXM1 for overall survival (OS) and relapse-free survival (RFS) in HPV-positive oral and lung cancers was assessed using Kaplan-Meier and Cox regression models. RESULTS: Herein, FOXM1 expression is upregulated by E6-mediated NKX2-1 in HPV-positive cervical, oral, and lung cancer cells. Induction of FOXM1 by E6 through the MZF1/NKX2-1 axis is responsible for HPV-mediated soft agar growth, invasiveness, and stemness through activating Wnt/β-catenin signaling pathway. In a nude mice model, metastatic lung tumor nodules in HPV 18 E6-positive GNM or HPV 16 E6-positive TL-1–injected nude mice were markedly decreased in both cell types with E6 knockdown, FOXM1 knockdown, or treatment with FOXM1 inhibitor (thiostrepton). Among the four subgroup patients, the worst FOXM1 prognostic value for OS and RFS was observed in HPV 16/18–positive patients with tumors with high-expressing FOXM1. CONCLUSIONS: Induction of FOXM1 by E6 oncoprotein through the MZF1/NKX2-1 axis may be responsible for HPV 16/18–mediated tumor progression and poor outcomes in HPV-positive patients.

Programmed death ligand (PD-L1) expression was associated with tumor immune escape and subsequent poor prognosis in non-small cell lung cancer (NSCLC). This expression was higher in patients with EGFR-mutated NSCLC tumors than in those with EGFR-wild-type (WT) NSCLC tumors. We therefore hypothesized that poor prognosis mediated by higher PD-L1 may be partially through conferring resistance to tyrosine kinase inhibitor (TKI) in NSCLC regardless of EGFR mutation. The change in PD-L1 expression following gene manipulation corresponded with changes in expression of HIF-1α and YAP1. The expression of HIF-1α and YAP1 was concomitantly decreased by PD-L1 silencing or by ROS scavenger treatment (N-acetylcysteine, NAC); however, a ROS inducer treatment (pyocyanin) completely reversed the decreased expression of both genes in EGFR-mutated and -wild-type (WT) NSCLC cells. The MTT assay indicated that the inhibitory concentration of gefitinib yielding 50% cell viability (IC50) depended on PD-L1-mediated YAP1 expression. Mechanistic studies indicated that upregulation of YAP1 by PD-L1 might be responsible for EGFR mutation-independent TKI resistance via the ROS/HIF-1α axis. An unfavorable TKI response was more common in patient tumors with high PD-L1 or YAP1 mRNA expression than in patient tumors with low mRNA expression of these genes. In conclusion, PD-L1 might confer EGFR mutation-independent TKI resistance in NSCLC cells via upregulation of YAP1 expression.

The inner thoracic cavity is lined by the parietal pleura, and the lung lobes are covered by the visceral pleura. The parietal and visceral plurae form the pleural cavity that has negative pressure within to enable normal respiration. The lung tissues are bilaterally innervated by vagal and spinal nerves, including sensory and motor components. This complicated innervation pattern has made it difficult to discern the vagal vs. spinal processes in the pulmonary visceral pleura. With and without vagotomy, we identified vagal nerve fibres and endings distributed extensively in the visceral pleura ('P'-type nerve endings) and triangular ligaments ('L'-type nerve endings) by injecting wheat germ agglutinin-horseradish peroxidase as a tracer into the nucleus of solitary tract or nodose ganglion of male Sprague-Dawley rats. We found the hilar and non-hilar vagal pulmonary pleural innervation pathways. In the hilar pathway, vagal sub-branches enter the hilum and follow the pleural sheet to give off the terminal arborizations. In the non-hilar pathway, vagal sub-branches run caudally along the oesophagus and either directly enter the ventral-middle-mediastinal left lobe or follow the triangular ligaments to enter the left and inferior lobe. Both vagi innervate: (i) the superior, middle and accessory lobes on the ventral surfaces that face the heart; (ii) the dorsal-rostral superior lobe; (iii) the dorsal-caudal left lobe; and (iv) the left triangular ligament. Innervated only by the left vagus is: (i) the ventral-rostral and dorsal-rostral left lobe via the hilar pathway; (ii) the ventral-middle-mediastinal left lobe and the dorsal accessory lobe that face the left lobe via the non-hilar pathway; and (iii) the ventral-rostral inferior lobe that faces the heart. Innervated only by the right vagus, via the non-hilar pathway, is: (i) the inferior (ventral and dorsal) and left (ventral only) lobe in the area near the triangular ligament; (ii) the dorsal-middle-mediastinal left lobe; and (iii) the right triangular ligament. Other regions innervated with unknown vagal pathways include: (i) the middle lobe that faces the superior and inferior lobe; (ii) the rostral-mediastinal inferior lobe that faces the middle lobe; and (iii) the ventral accessory lobe that faces the diaphragm. Our study demonstrated that most areas that face the dorsal thoracic cavity have no vagal innervation, whereas the interlobar and heart-facing areas are bilaterally or unilaterally innervated with a left-rostral vs. right-caudal lateralized innervation pattern. This innervation pattern may account for the fact that the respiratory regulation in rats has a lateralized right-side dominant pattern.

Chemical mechanical planarization ͑CMP͒ has played an enabling role in producing near-perfect planarity of interconnection and metal layers in ultralarge scale integrated devices. For stable and high performance of CMP, it is important to ensure uniform slurry flow at the pad-wafer interface, hence necessitating the use of grooved pads that help discharge debris and prevent subsequent particle loading effects. Here, using two-dimensional lubrication theory and contact mechanics models, we examine the effects of pad groove designs ͑viz. their width, depth, and spacing͒ on slurry flow in CMP. It is found that the presence of pad grooves generally increases the slurry flow rate ͑which clearly facilitates debris discharge͒ and the magnitude of the subambient fluid pressure ͑i.e., suction͒ on the pad-wafer interface. The increased suction implies higher contact stress on the pad-wafer interface, and hence the local material removal rate is expected to increase as well. However, our numerical results suggest that, as a grooved pad has less contact area for effective interaction with the wafer, the overall material removal rate is expected to increase as well. However, our numerical results suggest that, as a grooved pad has less contact area for effective interaction with the wafer, the overall material removal rate is decreased by the presence of pad grooves. There is therefore a trade-off between slurry flow rate enhancement and material removal rate reduction in pad groove design.Chemical mechanical planarization ͑CMP͒, also known as chemical mechanical polishing, is now widely recognized as the technology of choice for eliminating topographic variations and achieving near-perfect planarity of interconnection and metal layers in ultralarge scale integrated ͑ULSI͒ devices. 1 During the CMP process, a rotating wafer is pressed face down against a rotating pad, while a slurry containing chemicals and abrasive particles is dragged into the pad-wafer interface. Polishing is then accomplished by the interaction of the pad and slurry with the wafer surface or by direct contact between the wafer and pad. 2 To ensure stable and high performance of CMP, especially for wafers of larger diameters, it is important to optimize the slurry, pad, and other consumables. 3,4 This has thus necessitated the use of grooved pads that help discharge debris and prevent subsequent particle loading effects. 5,6 Through theoretical modeling and numerical calculations, this paper aims to understand the effects of pad grooves on slurry flow at the pad-wafer interface, and to gain some insights into the optimization of pad groove designs thereby. In particular, a theoretical model previously used by Shan et al. 7 is adapted to take into account the presence of grooves on the polishing pad. As a matter of fact, there have been a number of other theoretical models in the literature, 8-14 each considering certain aspects of the CMP process. Specifically, among the works related to the calculation of slurry flow for grooved pads, Subramanian et al. 1...

Magnesium superoxide dismutase (SOD2) has been shown to cause dysfunction of p53 transcriptional activity, whereas, in turn, SOD2 expression is regulated by p53 to modulate lung tumorigenesis. In this study, we found that the level of SOD2 expression in a panel of lung cancer cells was negatively correlated with that of NK2 homeobox 1 (NKX2-1) but was not associated with p53 status. Mechanistic studies indicated that a decrease in NKX2-1 caused by SOD2-activated IKKβ transcription was achieved by derepression of binding of Sp1 to the IKKβ promoter. Immunoprecipitation, glutathione S-transferase pull-down experiments and electrophoretic mobility shift assays demonstrated a direct interaction between NKX2-1 and Sp1, blocking Sp1-mediated IKKβ transcription. SOD2-mediated nuclear factor-kappaB activation, via elevation of IKKβ transcription, promoted anchorage-independent soft-agar growth, invasion and xenograft tumor formation, because of development of the epithelial-to-mesenchymal transition. The expression level of NKX2-1 messenger RNA was negatively associated with the extent of SOD immunostaining and the IKKβ messenger RNA expression level in lung tumors. The extent of SOD2 immunostaining and IKKβ messenger RNA levels may independently predict overall survival and relapse-free survival in lung adenocarcinoma patients. In summary, we found that SOD2 activates nuclear factor-kappaB signaling by increasing IKKβ transcription, which results in progression of lung adenocarcinoma and poorer patient outcomes. We suggest that IKKβ may potentially be targeted to improve outcomes in patients with SOD2-positive tumors.