Induced Lung Cancer Research Articles

Serglycin in tumor microenvironment promotes non-small cell lung cancer aggressiveness in a CD44-dependent manner

Tumor microenvironment (TME) plays an active role in promoting tumor progression. To further understand the communication between TME and tumor cells, this study aimed at investigating the involvement of CD44, a type I cell surface receptor, in the crosstalk between tumor cells and TME. We have previously shown that chondroitin sulfate proteoglycan serglycin (SRGN), a CD44-interacting factor, was preferentially secreted by cancer-associated fibroblasts (CAFs) for promoting tumor growth in breast cancer patients. In this study, we show that SRGN is overexpressed in primary non-small cell lung cancers (NSCLCs), by both carcinoma and stromal cells. Using gain-of-function and loss-of-function approaches, we show that SRGN promotes NSCLC cell migration and invasion as well as colonization in the lung and liver in a CD44-dependent manner. SRGN induces lung cancer cell stemness, as demonstrated by its ability to enhance NSCLC cell sphere formation via Nanog induction, accompanied with increased chemoresistance and anoikis-resistance. SRGN promotes epithelial-mesenchymal transition by enhancing vimentin expression via CD44/NF-κB/claudin-1 (CLDN1) axis. In support, CLDN1 and SRGN expression are tightly linked together in primary NSCLC. Most importantly, increased expression of SRGN and/or CLDN1 predicts poor prognosis in primary lung adenocarcinomas. In summary, we demonstrate that SRGN secreted by tumor cells and stromal components in the TME promotes malignant phenotypes through interacting with tumor cell receptor CD44, suggesting that a combined therapy targeting both CD44 and its ligands in the TME may be an attractive approach for cancer therapy.

Design, synthesis, and evaluation of asymmetric EF24 analogues as potential anti-cancer agents for lung cancer

The nuclear factor-kappa B (NF-κB) signaling pathway has been targeted for the therapy of various cancers, including lung cancer. EF24 was considered as a potent inhibitor of NF-κB signaling pathway. In this study, a series of asymmetric EF24 analogues were synthesized and evaluated for their anti-cancer activity against three lung cancer cell lines (A549, LLC, H1650). Most of the compounds exhibited good anti-tumor activity. Among them, compound 81 showed greater cytotoxicity than EF24. Compound 81 also possessed a potent anti-migration and anti-proliferative ability against A549 cells in a concentration-dependent manner. Moreover, compound 81 induced lung cancer cells death by inhibiting NF-κB signaling pathway, and activated the JNK-mitochondrial apoptotic pathway by increasing reactive oxygen species (ROS) generation resulting in apoptosis. In summary, compound 81 is a valuable candidate for anti-lung cancer therapy.

Reduced apurinic/apyrimidinic endonuclease activity enhances the antitumor activity of oxymatrine in lung cancer cells.

Lung cancer is the leading cause of cancer-related deaths worldwide and is associated with a very poor outcome. Oxymatrine exerts antitumor effects by inducing apoptosis and inhibiting the proliferation of different cancer cells; however, the anticancer effects and mechanism of action of oxymatrine have not been evaluated sufficiently in human lung cancer cells. Thus, the present study aimed to investigate the anticancer effects of oxymatrine in human lung cancer cells and identify the molecular mechanisms underlying these effects. MTT assays demonstrated that oxymatrine significantly inhibited the proliferation of A549 and H1299 cells in a time- and dose-dependent manner. In addition, flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assays suggested that oxymatrine treatment may induce lung cancer cell apoptosis in a dose-dependent manner. Furthermore, we detected that oxymatrine induced a significant increase in DNA damage and the expression of PARP and phosphorylated H2AX, and a significant decrease in that of nuclear APE1 and AP endonuclease activity in A549 cells. APE1 knockdown cells (APE1shRNA) plus oxymatrine treatment reduced cells proliferation and induced apoptosis more seriously than control shRNA cells. This appeared to be a consequence of an increase in the number of apurinic/apyrimidinic (AP) sites, DNA damage, PARP and H2AX phosphorylation, which together resulted in the induction of apoptosis. In contrast, the sensitizing effects of APE1 overexpression plus oxymatrine treatment did not occur in APEOE cells. These findings reveal a potential mechanism of action for oxymatrine-induced apoptosis and suggest that oxymatrine is a promising potential therapeutic agent for the treatment of lung cancer.

Osthole induces lung cancer cell apoptosis through inhibition of inhibitor of apoptosis family proteins.

In the present study, we investigated the effects and mechanisms of Osthole on the apoptosis of non-small cell lung cancer (NSCLC) cells and its synergistic effect with Embelin. Our results revealed that treatment with both Osthole and Embelin inhibited cell proliferation. Notably, combination treatment of Osthole and Embelin inhibited cell proliferation more significantly compared with monotherapy. In addition, morphological analysis and Annexin V/propidium iodide analysis revealed that the combination of Osthole and Embelin enhanced their effect on cell apoptosis. We further examined the effect of Osthole on the expression of inhibitor of apoptosis protein (IAP) family proteins. That treatment of A549 lung cancer cells with various concentrations of Osthole was observed to decrease the protein expression of X-chromosome-encoded IAP, c-IAP1, c-IAP2 and Survivin, and increase Smac expression in a dose-dependent manner. Furthermore, it was noted that Osthole or Embelin alone increased the expression of BAX, caspase-3, caspase-9, cleaved caspase-3 and cleaved caspase-9, and decreased Bcl-2 levels following treatment. Osthole and Embelin combination treatment had a synergistic effect on the regulation of these proteins. In conclusion, our study demonstrated that Osthole inhibited proliferation and induced the apoptosis of lung cancer cells via IAP family proteins in a dose-dependent manner. Osthole enhances the antitumor effect of Embelin, indicating that combination of Osthole and Embelin has potential clinical significance in the treatment of NSCLC.

Detection of Helicobacter pylori in the Bronchoalveolar Lavage of Patients with Lung Cancer Using Real-Time PCR.

BackgroundLung cancer is one of the most common causes of death worldwide. Although smoking and environmental pollutants are the most important risk factors of lung cancer, the role of infectious causes should also be considered in the pathogenesis and progress of lung cancer.ObjectivesThis study examined the relationship between Helicobacter pylori and lung cancer through serology, real-time PCR, and urease tests.MethodsThis descriptive cross-sectional study was conducted on 52 adult patients with lung cancer who were selected after having their history taken and being physically examined by a pulmonologist. Then, the patients underwent a bronchoscopy, a BAL, and biopsy sampling. A urease test was run for each biopsy sample, real-time PCR was used for each BAL sample, and H. pylori serology was used for each patient’s serum.ResultsThe patients’ average age was 60.65 ± 9.15 years; 11.5% were female and 88.5% were male. The prevalence of H. pylori in lung cancer patients was 11.5% according to the BAL PCR test, 92.3% according to the serology test, and 3.8% according to the urease test.ConclusionsThe results demonstrated an association between of lung cancer and H. Pylori infection via the hypothesis of direct damage and chronic inflammation through inhalation and aspiration and the systematic immune response induced by H. pylori colonization. Helicobacter pylori, together with a host’s genetic predisposition and other environmental risk factors, could be attributed to the induction of lung cancer.

Risk evaluation in patients undergoing coronary angiographic and angioplastic procedures with radial access

Introduction Nowadays interventional cardiac procedures are of concern for the relatively high doses delivered to patients. Purpose To estimate the effective dose (E), the organ doses (H t ) and the related risk for cancer induction in terms of radiation exposure-induced death (REID) in patients undergoing coronary angiography (CA) and percutaneous transluminal coronary angioplasty (PTCA) procedures with radial access. Materials and methods 65 patients underwent CA and PTCA interventions on a Philips Allura XPer FD10 angiographic equipment. For each patient, Xray tube voltage, projection, filtration, field dimensions and DAP were obtained by filming the monitor of the workstation during the entire procedure. E and H t were evaluated by means of the MonteCarlo code PCXMC that models radiation beam and transport in an anthropomorphic phantom, allowing also the evaluation of REID. Results Mean fluoroscopy time, DAP and E were: 3.1 min, 29.9 Gyxcm 2 , 9.1 mSv and 8.9 min, 60.9 Gyxcm 2 , 20.3 mSv for CA and PTCA, respectively; the correlation with DAP was very good in CA (r = 0.99) and in PTCA (r = 0.93). For both CA and PTCA, the most irradiated organs were lungs, oesophagus and red marrow, whose mean H t were 32.5 mSv, 30.7 mSv, 11.6 mSv for CA and 58.5 mSv, 66.7 mSv, 24.9 mSv for PTCA; the highest REIDs were for induction of leukemia (0.04% and 0.08%) and lung cancer (0.14% and 0.20%). Conclusions Cardiac interventional procedures with radial access improves patient compliance, reduces haemorrhagic complications compared to femoral access with comparable E and Ht values.

High resolution metabolomics to discriminate compounds in serum of male lung cancer patients in South Korea

BackgroundThe cancer death rate escalated during 20th century. In South Korea, lung cancer is expected to contribute 12,736 deaths in men, the highest amount among all cancers. Several risk factors may increase the chance to acquiring lung cancer, with mostly related to exogenous compounds found in cigarette smoke and synthetic manufacturing materials. As the mortality rate of lung cancer increases, deeper understanding is necessary to explore risk factors that may lead to this malignancy. In this regard, this study aims to apply high resolution metabolomics (HRM) using LC-MS to detect significant compounds that might contribute in inducing lung cancer and find the correlation of these compounds to the subjects’ smoking habit.MethodsThe comparison was made between healthy control and lung cancer groups for metabolic differences. Further analyses to determine if these differences are related to tobacco-induced lung cancer (past-smoker control vs. past-smoker lung cancer patients (LCPs) and non-smoker control vs. current-smoker LCPs) were selected. The univariate analysis was performed, including a false discovery rate (FDR) of q = 0.05, to determine the significant metabolites between the analyses. Hierarchical clustering analysis (HCA) was done to discriminate metabolites between the control and case subjects. Selected compounds based on significant m/z features of human serum then experienced MS/MS examination, showing that for many m/z, the patterns of ion dissociation matched with standards. Then, the significant metabolites were identified using Metlin database and features were mapped on the human metabolic pathway mapping tool of the Kyoto Encyclopedia of Genes and Genomes (KEGG).ResultsUsing metabolomics-wide association studies, metabolic changes were observed among control group and lung cancer patients. Bisphenol A (211.11, [M + H-H2O]+), retinol (287.23, [M + H]+) and L-proline (116.07, [M + H]+) were among the significant compounds found to have contributed in the discrimination between these groups, suggesting that these compounds might be related in the development of lung cancer. Retinol has been seen to have a correlation with smoking while both bisphenol A and L-proline were found to be unrelated.ConclusionsTwo potential biomarkers, retinol and L-proline, were identified and these findings may create opportunities for the development of new lung cancer diagnostic tools.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-016-0419-3) contains supplementary material, which is available to authorized users.

Abstract 3655: Suppression of Plk3 expression by nickel may contribute to lung carcinogenesis

Abstract Exposure to nickel (Ni) compounds is known to induce human lung malignancies. Research shows that Ni induces lung cancer mainly through epigenetic and metabolic pathways. Polo-like kinase 3 (Plk3) is a multifunctional protein kinase that involves in tumor initiation and progression. Plk3−/− mice tend to develop highly vascularized lung tumors at an advanced age. Reduced expression of Plk3 was reported in human lung cancers. In this study, we examined the effect of Ni on Plk3 expression in A549 lung carcinoma cells. Western blot showed that NiCl2 reduced the Plk3 protein level in a time- and dose-dependent manner. We then treated A549 cells with cycloheximide for various times to examine the effect of NiCl2 on the half-life of Plk3 protein. We observed a shortened half-life of Plk3 in the presence of NiCl2. A reduced level of Plk3 protein was also found in hypoxia-treated A549 cells. In addition, MG132, a specific proteasome inhibitor, could partially reverse the effect of NiCl2 or hypoxia on the Plk3 protein level. Our cell fractionation experiments showed that majority of the endogenous Plk3 protein is located in the cytoplasm where degradation of Plk3 likely occurs. Transfection of A549 cells with FLAG-tagged ubiquitin (FLAG-Ub) followed by treatment with NiCl2 and/or MG132, immunoprecipitation (IP) with an anti-FLAG antibody, and Western blot with an anti-Plk3 antibody showed that Plk3 was poly-ubiquitinated and that the ubiquitination was significantly enhanced by NiCl2 and/or MG132 treatments. The elevated ubiquitination of Plk3 in response to NiCl2 was further confirmed in 293T cells with co-transfection of Plk3 and FLAG-Ub. Furthermore, overexpression of USP28, a deubiquitinase known to be inhibited by Ni or hypoxia, partially restored the protein level of Plk3 suppressed by NiCl2. Moreover, real-time PCR analysis showed that NiCl2 also reduced the Plk3 mRNA level, in a time- and dose-dependent pattern. Taken together, our data show that NiCl2 suppresses Plk3 expressions by lowering the Plk3 mRNA level and promoting proteasome-mediated degradation of the Plk3 protein. Given the potential role of Plk3 as a tumor suppressor in the lung, suppression of Plk3 expression likely contributes to Ni-induced lung carcinogenesis and tumor progression. Citation Format: Cen Li, Soyoung Park, Wei Dai, Dazhong Xu. Suppression of Plk3 expression by nickel may contribute to lung carcinogenesis. [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 3655.

Abstract 4398: Impact of Interleukin-22 on K-ras mutant lung tumor microenvironment and stemness properties

Abstract Oncogenic K-ras mutations found in ∼ 30% of all non-small cell lung cancers are associated with chemoresistance and poor prognosis. Using a K-ras induced lung cancer mouse model, CC-LR, we previously showed that K-ras mutant lung tumors have intrinsic inflammatory characteristics with activation of NF-kB pathway, release of inflammatory cytokines IL-6, and activation of the IL-6 responsive transcription factor STAT3. We have further shown that IL-6/STAT3 pathway, and IL-17 producing CD4 helper T cells (Th17 cells) through their main cytokine, IL-17A, play critical roles in promotion of lung cancer in this model. IL-22 is another effecter molecule secreted by Th17 cells which is highly expressed in our K-ras mutant mouse model. IL-22 is a unique cytokine, which seems to act exclusively on nonhematopoietic cells, with basal IL-22R expression in the epithelial cells and fibroblast, and mostly signals through STAT3 pathway. Here we found that genetic ablation of IL-22 in CC-LR mice (CC-LR/IL22-KO mice), causes significant reduction in lung surface tumor numbers by ∼54% (2.1-fold). Histopathological analysis of lung sections confirmed a reduction in number and size of tumors in CC-LR/IL22-KO mice, which was associated with significantly lower tumor cell proliferation, angiogenesis and STAT3 activation. IL-22 ablation also reduced the numbers of inflammatory cells in bronchoalveolar lavage fluid, and decreased the expression of pro-tumor inflammatory cytokines such as IL-6, IL-17 and TNFα. This was associated with increased expression of anti-tumor Th1 cells -specific transcription factor (Tbet) and their activation markers, IFNγ, and GZB, and decreased expression of pro-tumor Th17- (RORγ) and T regulatory (FOXP3+) specific transcription factors. Recent studies have shown an association between IL-22 and stem-cell like properties in colon cancer. In lung cancer, cell populations expressing NANOG, SOX2, Oct4 and/or aldehyde dehydrogenase activity are enriched with stemness properties. Interestingly, in CC-LR/IL22-KO mice we found significant reduction in expression of these stemness genes. Thus, we conclude that IL-22 promotes K-ras mutant lung tumorigenesis by inducing a pro-tumor inflammatory microenvironment with proliferative and angiogenic properties as well as protecting stemness characteristic in epithelial/tumor cells. Therefore, we propose pharmacological targeting of IL-22 as a potential therapeutic strategy in combination with conventional cytotoxic therapy, immune check point blockade, or other targeted therapies (e.g. MEK inhibition) for lung cancer patients with K-ras mutation. Citation Format: Nasim Khosravi, Amber M. Cumpian, Soudabeh Daliri, Cynthia De La Garza, Mauricio S. Caetano, Seyed Javad Moghaddam. Impact of Interleukin-22 on K-ras mutant lung tumor microenvironment and stemness properties. [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 4398.

Apple Cider Vinegar (A Prophetic Medicine Remedy) Protects against Nicotine Hepatotoxicity: A Histopathological and Biochemical Report

Nicotine is the most abundant component in cigarette smoking and is involved in the pathogenesis of lung cancer and increases the risk of developing hepatocellular carcinoma and liver cirrhosis. Prevention of nicotine- induced lung cancer and liver damage may be achieved via decreasing nicotine-induced pathological effects. Nicotine is metabolized in the liver. Natural diet contains a variety of compounds e.g. apple cider (ACV) that exhibits protective effects against different toxins. This study aims to investigate the effects of nicotine on the liver using morphometrical, histopathological and biochemical parameters and study the protective effect of ACV against toxicity of nicotine. Three groups of the male albino rat were used: untreated control group, nicotine treated group (4 mg/kg/day) while the third group received both ACV (2ml/kg/day) and nicotine (4 mg/kg/day). Treatment was given for 30 days. There was a significant increase in the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) together with a damage and degeneration in the liver tissues of the nicotine treated groups. ACV administration to nicotine-treated rats showed near normal liver biochemical markers with reduction in the tissue damage associated with the nicotine administration. ACV administration to nicotine-treated rat ameliorated the decrease in the size of the hepatocytes nuclei. These results, along with previous observations, suggest that ACV may be useful in combating tissue injury resulting from nicotine toxicity. In prophetic medicine, Prophet Muhammad peace be upon him strongly recommended eating in the prophetic hadeeth: vinegar is the best edible. Conclusion: These finding confirm that chronic nicotine administration causes harmful effects to the liver and suggest that ACV may be useful in combating tissue injury resulting from nicotine toxicity. Hence, the intake of ACV might suppress the toxicity and mutagenic activity of nicotine. ACV may protect against nicotine-induced carcinogenesis.

Gefitinib induces lung cancer cell autophagy and apoptosis via blockade of the PI3K/AKT/mTOR pathway.

Gefitinib is a selective inhibitor of the tyrosine kinase epidermal growth factor receptor, which inhibits tumor pathogenesis, metastasis and angiogenesis, as well as promoting apoptosis. Therefore, gefitinib presents an effective drug for the targeted therapy of lung cancer. However, the underlying mechanisms by which gefitinib induces lung cancer cell death remain unclear. To investigate the effects of gefitinib on lung cancer cells and the mechanism of such, the present study analyzed the effect of gefitinib on the autophagy, apoptosis and proliferation of the A549 and A549-gefitinib-resistant (GR) cell lines GR. The regulation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/ mammalian target of rapamycin (mTOR) pathway was also investigated. Acridine orange staining revealed that gefitinib induced autophagy of A549 cells but not A549-GR cells. In addition, gefitinib promoted apoptosis and inhibited proliferation of A549 cells but not A549-GR cells. Furthermore, western blot analysis demonstrated that gefitinib treatment led to the downregulation of PI3K, AKT, pAKT, mTOR and phosphorylated-mTOR protein expression in A549 cells but not A549-GR cells. LY294002 blocked the PI3K/AKT/mTOR pathway and induced autophagy and apoptosis of A549 cells, however, no synergistic effect was observed following combined treatment with gefitinib and LY294002. In conclusion, the results of the present study indicate that gefitinib promotes autophagy and apoptosis of lung cancer cells via blockade of the PI3K/AKT/mTOR pathway, which leads to lung cancer cell death.

Secondhand Smoke Enhances Lung Cancer Risk in Male Smokers: An Interaction.

Previous studies revealed that some indoor air pollutants and fine particle matter can interact with active smoking, enhancing lung cancer risk in smokers. Secondhand smoke (SHS), with remarkable differences from active smoking, contributes significantly to indoor air pollution and generates a considerable amount of fine particle matter, may cause a similar interaction with active smoking. Information on lifetime SHS along with active smoking and other confirmed or suspected risk factors for lung cancer was collected in this case-referent study. Odds ratios and the 95% confidence intervals (95% CIs) of smoking status in different levels of SHS were evaluated. Potential multiplicative and additive interactions were explored. Compared with never-smokers without SHS, current smokers who were exposed to a high level of SHS demonstrated the highest odds ratio (15.13, 95% CI: 8.60, 26.65), almost doubles the effect in the current smokers without SHS. Significant additive interactions between current smoking and high level of SHS were observed for all lung cancers (synergy index = 1.80, 95% CI: 1.02, 3.24) and the squamous carcinoma subgroup. High level of SHS exposure greatly enhanced lung cancer risk among current smokers, consistent with an additive interaction; while this interaction was predominant for the squamous carcinoma. The results provide new evidence to the rationale of promoting global smoking cessation. Some indoor air pollutants can interact with active smoking, yielding a synergistic effect on inducing lung cancer. SHS, with noticeable differences from active smoking, is a major source of indoor air pollution. However, little has been known about the effect of SHS in smokers and whether there is a similar interaction between SHS and active smoking. In this study, we evaluated their separate and joint effects and indeed found a more than additive interaction between them. This finding suggests a potential problem of gathering smoking aggravating by venue restriction policies and re-advocates policy efforts on smoking cessation.

Nicotine Induced Lung Cancer Cells Epithelial-mesenchymal Transition and Promote Its Vitro Invasion Potential

背景与目的我们的前期研究发现尼古丁能诱导肺癌细胞上皮间质转化。本研究的目的是探讨尼古丁诱导的上皮间质转化（epithelial-mesenchymal transition, EMT）与肺癌侵袭之间的关系。方法应用不同浓度尼古丁处理肺腺癌A549细胞，应用Real-time PCR和Western blot方法检测EMT相关分子标志物E-钙粘蛋白（E-cadherin）和波形蛋白（Vimentin）mRNA和蛋白表达水平，应用免疫荧光技术检测β-链蛋白（β-catenin）蛋白表达位置的变化，应用划痕实验和Transwell小室侵袭实验检测尼古丁对肺癌细胞迁移侵袭能力的影响。结果尼古丁明显下调肺癌细胞株A549 E-cadherin mRNA和蛋白水平表达（P < 0.01, P < 0.01），并具有浓度和时间依赖性；尼古丁明显上调肺癌细胞株A549 Vimentin mRNA和蛋白水平表达（P < 0.01, P < 0.01）；尼古丁诱导肺癌细胞株A549细胞β-catenin蛋白发生核转移；划痕实验和侵袭实验观察到尼古丁处理的肺癌细胞株A549细胞的迁移和侵袭能力明显增强（P < 0.01, P < 0.01）。结论尼古丁能够诱导肺癌细胞发生EMT，并且促进肺癌细胞株A549细胞的体外侵袭潜能。

55P Development of a nanoformulation containing ganoderic acid for the prevention and treatment of the benzo[a]pyrene induced lung cancer

55P Development of a nanoformulation containing ganoderic acid for the prevention and treatment of the benzo[a]pyrene induced lung cancer

Nm23‐mediated hnRNPA2/B1 to regulate Sp1 expression is associated with lung cancer progression

Recent studies have indicated that decrease in Sp1 level is benefit to the lung cancer malignancy. In this study, we found that Nm23 not only increased phosphorylation of Sp1 at Thr739 to enhance Sp1 protein stability but also formed a complex with hnRNPA2/B1 that was recruited to the 5′‐UTR of Sp1 mRNA. Knocking down Nm23 or hnRNPA2/B1 decreased Sp1 expression through inhibiting cap‐independent translation manner and also increased the migratory activity of lung cancer cell lines. Furthermore, lung cancer patients with poor prognosis had low levels of Sp1 and Nm23, suggesting association between Nm23/Sp1 levels and survival rate. Studies performed to elucidate the mechanism underlying this relationship indicated that a decrease in Nm23 levels inducing lung cancer cells malignancy and inhibited hnRNPA2/B1 protein stability, and thus subsequently decreased the recruitment of hnRNPA2/B1 to the 5′‐UTR of Sp1 mRNA, repressing Sp1 expression through inhibition of the cap‐independent transcriptional activity. Overexpression of Sp1 abolished hnRNPA2/B1‐induced F‐actin polymerization and upregulation of epithelial‐mesenchymal transition (EMT)‐markers such as N‐cadherin, suggesting that hnRNPA2/B1‐mediated Sp1 expression is critical for lung cancer progression. Taken together, these results suggest that understanding the relationship between Nm23, hnRNPA2/B1, and Sp1 in regulating lung cancer malignancy will be beneficial of lung cancer.

Nanopharmaceutical Approach for Enhanced Anti-cancer Activity of Betulinic Acid in Lung-cancer Treatment via Activation of PARP: Interaction with DNA as a Target: -Anti-cancer Potential of Nano-betulinic Acid in Lung Cancer.

Objectives:This study examined the relative efficacies of a derivative of betulinic acid (dBA) and its poly (lactide- co-glycolide) (PLGA) nano-encapsulated form in A549 lung cancer cells in vivo and in co-mutagen [sodium arsenite (SA) + benzo]undefined[a]pyrene (BaP)]-induced lung cancer in mice in vivo.Methods:dBA was loaded with PLGA nanoparticles by using the standard solvent displacement method. The sizes and morphologies of nano-dBA (NdBA) were determined by using transmission electron microscopy (TEM), and their intracellular localization was verified by using confocal microscopy. The binding and interaction of NdBA with calf thymus deoxyribonucleic acid (CT-DNA) as a target were analyzed by using conventional circular dichroism (CD) and melting temperature (Tm) profile data. Apoptotic signalling cascades in vitro and in vivo were studied by using an enzyme-linked immunosorbent assay (ELISA); the ability of NdBA to cross the blood-brain barrier (BBB) was also examined. The stage of cell cycle arrest was confirmed by using a fluorescence-activated cell-sorting (FACS) data analysis.Results:The average size of the nanoparticles was ~ 110 nm. Confocal microscopy images confirmed the presence of NdBA in the cellular cytoplasm. The bio-physical properties of dBA and NdBA ascertained from the CD and the Tm profiles revealed that NdBA had greater interaction with the target DNA than dBA did. Both dBA and NdBA arrested cell proliferation at G0/G1, NdBA showing the greater effect. NdBA also induced a greater degree of cytotoxicity in A549 cells, but it had an insignificant cytotoxic effect in normal L6 cells. The results of flow cytometric, cytogenetial and histopathological studies in mice revealed that NdBA caused less nuclear condensation and DNA damage than dBA did. TEM images showed the presence of NdBA in brain samples of NdBA fed mice, indicating its ability to cross the BBB.Conclusion:Thus, compared to dBA, NdBA appears to have greater chemoprotective potential against lung cancer.

AKT1E¹⁷K Is Oncogenic in Mouse Lung and Cooperates with Chemical Carcinogens in Inducing Lung Cancer.

The hotspot AKT1E17K mutation in the pleckstrin homology domain of AKT1 occurs in approximately 0.6–2% of human lung cancers. Recently, we have demonstrated that AKT1E17K transforms immortalized human bronchial cells. Here by use of a transgenic Cre-inducible murine strain in the wild type Rosa26 (R26) locus (R26-AKT1E17K mice) we demonstrate that AKT1E17K is a bona-fide oncogene and plays a role in the development of lung cancer in vivo. In fact, we report that mutant AKT1E17K induces bronchial and/or bronchiolar hyperplastic lesions in murine lung epithelium, which progress to frank carcinoma at very low frequency, and accelerates tumor formation induced by chemical carcinogens. In conclusion, AKT1E17K induces hyperplasia of mouse lung epithelium in vivo and cooperates with urethane to induce the fully malignant phenotype.

Tree shrew as a new animal model for the study of lung cancer.

Animal models play a key role in identifying treatments for various types of cancer, including lung cancer. The aim of the present study was to develop a new animal model for lung cancer induction using tree shrews from the Yunnan region in China. Tree shrews are suitable for a full simulation of human disease because their structure, function and metabolism are adequately close to human. This animal may offer a new experimental animal model to be used in the study of lung cancer. In the present study, 80 healthy tree shrews were distributed in experimental and control groups. Animals in the experimental group received different concentrations of iodized oil suspension of 3-methylcholanthrene (3-MC) and diethylnitrosamine (DEN) while animals in the control groups received saline or lipiodol solvent via endotracheal instillation. In the 3rd, 5th, 7th, 9th and 11th weeks the body weights of the animals were measured and chest X-ray examinations were conducted. Pathological studies on the lung tissues were also performed and the pathological changes occurring in bronchial epithelium in all the groups were examined. Animals in the experimental group gradually lost their body weight. For tree shrews in the blank control and solvent control groups the survival rates were 100 and 80%, respectively while the survival rate for the experimental group was 0%. Results from the chest X-ray conducted on animals in the blank control and solvent control groups revealed no obvious abnormalities while in the experimental group high-density shadow spots within the perfusion sites were observed. Pathological studies performed on these high-density areas confirmed changes in the bronchial epithelium. In the experimental groups we also detected bronchial epithelial atypical hyperplasia, and apparent changes in carcinoma in situ. In conclusion, lung cancer was successfully induced in tree shrews by a one-time endotracheal introduction of iodized oil suspension of 3-MC and DEN.

EGCG induces lung cancer A549 cell apoptosis by regulating Ku70 acetylation.

Lung cancer is the leading cause of cancer-related death worldwide. (-)-Epigallocatechin-3-gallate (EGCG) is a potential chemopreventive and therapeutic agent for lung cancer. Induction of apoptosis was examined using Annexin V/PI double staining flow cytometry. Western blot analysis detected the protein expression of cleaved caspase-3, Bax and Bcl-xL. Co-immunoprecipitation was used to detect the interaction of Ku70-Bax and the acetylation status of Ku70. Treatment of A549 cells with EGCG-induced apoptosis via increased expression of cleaved caspase-3 and Bax, but decreased expression of Bcl-xL. EGCG upregulated the K70 acetylation status of A549 cells and downregulated the interaction of Bax-Ku70 in a concentration- and time-dependent manner. The apoptosis-promoting effect of EGCG on A549 cells was obviously weakened, along with strengthening of the Bax-Ku70 interaction, after pCDNA3.1(+)-Ku70 plasmid and pCDNA3.1(+)-Ku70539/542R plasmid transfection. Our results established a role of EGCG in inducing cell apoptosis by suppressing Bax activity. Regulating Ku70 acetylation by EGCG, that block the interaction between Ku70 and Bax, will result in lung cancer cell apoptosis.

Abstract B120: Characterization and combination immunotherapy treatment of an inducible autochthonous murine lung cancer model expressing human carcinoembryonic antigen (CEA) as a tumor-associated self-antigen

Abstract Previous work from our lab has demonstrated that a combination of anti-tumor antibody and an IL-2 fusion protein that exhibits extended serum half-life elicits an immune response that can effectively control a wide variety of tumor models. We have since then combined this therapeutic regimen with a vaccine exhibiting efficient lymph node trafficking that can generate an impressive population of tumor-antigen specific CD8+ T-cells but by itself does not provide good anti-tumor efficacy. The efficacy of this combination immunotherapy is further boosted by immune checkpoint blockade, leading to a robust four-component therapy: 1) anti-tumor antigen antibody; 2) IL-2 fusion protein; 3) anti-tumor antigen vaccine; 4) anti-PD-1 antibody. Although this four-pronged approach is demonstrably effective in the syngeneic subcutaneous melanoma model B16F10, we wish to test its efficacy in a more physiological model. To this end, we have turned to a model developed by the Jacks Lab, known as the KP model: an inducible lung tumor model where lentivirus-driven integration and expression of Cre is able to activate oncogenic Kras and completely remove p53 function. Because our therapeutic regimen requires a targetable tumor-associated antigen with respect to both the antibody and vaccine, we chose to induce expression of human carcinoembryonic antigen (CEA) in these tumors, as CEA has a well-studied structure and biology, and frequently expresses aberrantly in many forms of human adenocarcinomas. Additionally, our lab has previously engineered an antibody targeting CEA possessing picomolar affinity. Finally, to remove any endogenous immunological response against human CEA as a foreign antigen in our mouse system, we have crossed the KP model with a mouse model transgenic for human CEA, which in the literature has been described to have identical spatiotemporal expression of CEA as found in humans and should allow for central tolerance of this antigen. In the course of this work, we have successfully introduced human CEA into our lentivirus constructs and shown tumorigenesis by these constructs in the KP model coincides with expression of tumor-associated CEA, as detected by qPCR. On the therapeutic side, we have tailored the vaccine to successfully drive an anti-CEA CD8+ T-cell response. Performing preliminary therapeutic experiments in a transplant model of the KP tumor with our four-component therapy, we saw tumor control compared to untreated tumors. Upon interrogating the CD8+ T-cell response against CEA, we found 1-15% of CD8+ T-cells in the blood respond to CEA stimulation by intracellular cytokine staining. With regards to the lung tumor model, in the course of establishing the system we have also observed that the growth kinetics of tumors expressing CEA lags behind those tumors without CEA, even in the transgenic background. Preliminary immunophenotyping work by flow cytometry suggests that tumors with CEA seem to have a reduced myeloid-derived suppressor cell (MDSC) population and a higher CD8a+ dendritic cell (DC) population compared to tumors without CEA, suggesting that the former may have a less immunosuppressive tumor microenvironment that is better able to prime an anti-tumor CD8+ T-cell response. We will be planning to conduct therapeutic trials in the more physiological lung tumor KP model in the near future, as well as investigate the differences in the immune response with tumors expressing or lacking CEA. Citation Format: Eric F. Zhu, Kavya Rakhra, Naveen Mehta, Kelly D. Moynihan, Cary F. Opel, Darrell J. Irvine, K. Dane Wittrup. Characterization and combination immunotherapy treatment of an inducible autochthonous murine lung cancer model expressing human carcinoembryonic antigen (CEA) as a tumor-associated self-antigen. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B120.