Urethane-induced Lung Tumors Research Articles

Anthocyanins prevent the development and progression of urethane-induced lung cancer by regulating energy metabolism in mice.

Anthocyanin (ACN) is a natural antioxidant with multiple biological activities, and the aim of this study was to evaluate the protective effect of ACN on the development and progression of lung cancer and to further explore its possible mechanism of action. In vivo, we fed C57BL/6J mice a 0.5%ACN diet or a control diet to observe their effects on the development and progression of urethane-induced lung cancer. In vitro, multiple lung cancer cell lines were used to investigate the effects of C3G on cell viability. The results showed a reduction in lung tumor burden and downregulation of oxidative phosphorylation and fatty acid degradation pathways in lung tissue of urethane-administrated ACN-fed mice compared with control diet-fed mice. In vitro, cyanidin-3-O-glucoside chloride (C3G) intervention treatment significantly inhibited proliferation and apoptosis of A549 cells. This process is likely due to the modulation of AMPK/mTOR signaling pathway by C3G to regulate cellular fatty acid metabolism and reduce intracellular lipid accumulation which affects the growth of lung cancer cells. These results suggest that ACN can inhibit the development and progression of urethane-induced lung tumors and alter the lipid metabolism of tumors in C57BL/6J mice.

NCAPG promotes the oncogenesis and progression of non-small cell lung cancer cells through upregulating LGALS1 expression

BackgroundNumerous common oncogenic driver events have been confirmed in non-small cell lung cancer (NSCLC). Although targeted therapy has revolutionized NSCLC treatment, some patients still do not respond. NCAPG, also known as non-SMC condensin I complex subunit G, was positively associated with proliferation and migration in several tumor types.MethodsWe used transcriptional sequencing and TCGA database analysis to identify NCAPG as a new therapeutic target for NSCLC. The oncogenic roles of NCAPG in NSCLC tumor growth and metastasis were detected in vitro and in vivo. Ncapg+/+ or Ncapg+/− mice with urethane treatment were analyzed for oncogenesis of NSCLC.ResultsWe investigated NCAPG as a new oncogenic driver which promoted NSCLC tumorigenesis and progression. We used transcriptome sequencing and the Cancer Genome Atlas (TCGA) database analysis to screen and found that NCAPG was negatively correlated with NSCLC survival. Using immunohistochemistry, we demonstrated that NCAPG overexpression was an independent risk factor for NSCLC survival. Functionally, NCAPG knockdown inhibited proliferation, migration, and invasion of NSCLC cells in vitro and in vivo. We exposed wildtype or Ncapg+/− mice to urethane and discovered that urethane-induced lung tumors were reduced in Ncapg+/− mice. Mechanistically, the function of NCAPG in promoting initiation and progression of NSCLC was closely related to LGALS1, which was also upregulated in NSCLC and might interact directly with NCAPG.ConclusionsThis study indicates that NCAPG is one of the essential factors for NSCLC oncogenesis and progression, providing a new target for prognosis prediction and treatment of NSCLC.

Overexpression of PRDX4 Modulates Tumor Microenvironment and Promotes Urethane-Induced Lung Tumorigenesis.

Peroxiredoxin 4 (PRDX4), initially reported as an antioxidant, is overexpressed in lung cancer and participates in its progression. However, its role in the urethane-induced lung tumor model is undetermined. The aim of this study was to investigate the effect of PRDX4 overexpression on carcinogen-induced lung tumor development. Human PRDX4 overexpression transgenic (Tg) mice (hPRDX4+/+) and non-Tg mice were intraperitoneally injected with urethane to induce lung tumor. After 6 months, tumor formation was compared between groups and possible mechanisms for the difference in tumor development were investigated. The serum and lung PRDX4 expressions were enhanced after urethane stimulation in Tg mice. Both the average number of tumors (≥0.5 mm) and tumor diameter per mouse in the Tg group were significantly larger than in non-Tg controls, while body weight was lower in the Tg group. Compared with non-Tg controls, tumor cell proliferation was enhanced, while tumor cell apoptosis was suppressed in Tg mice. Systemic oxidative stress and oxidative stress in lung tumors were inhibited by PRDX4 overexpression. The balance of prooxidant enzymes and antioxidant enzymes was also shifted to a decreased level in Tg tumor. In lung tumor tissue, the density of microvessel penetrated into tumor was higher in the Tg group; macrophage infiltration was enhanced in Tg tumors, while there was no difference in T lymphocyte infiltration; the expressions of cytokines, including interleukin-1 beta (IL-1β) and matrix metallopeptidase 9 (MMP9), were elevated in Tg tumors, which resulted from enhanced phosphorylation of nuclear factor-κB p65 (NF-κB p65) and c-Jun, respectively. In conclusion, PRDX4 overexpression modulated tumor microenvironment and promoted tumor development in the mouse urethane-induced lung cancer model.

K-Ras-Activated Cells Can Develop into Lung Tumors When Runx3-Mediated Tumor Suppressor Pathways Are Abrogated.

K-RAS is frequently mutated in human lung adenocarcinomas (ADCs), and the p53 pathway plays a central role in cellular defense against oncogenic K-RAS mutation. However, in mouse lung cancer models, oncogenic K-Ras mutation alone can induce ADCs without p53 mutation, and loss of p53 does not have a significant impact on early K-Ras–induced lung tumorigenesis. These results raise the question of how K-Ras–activated cells evade oncogene surveillance mechanisms and develop into lung ADCs. RUNX3 plays a key role at the restriction (R)-point, which governs multiple tumor suppressor pathways including the p14ARF–p53 pathway. In this study, we found that K-Ras activation in a very limited number of cells, alone or in combination with p53 inactivation, failed to induce any pathologic lesions for up to 1 year. By contrast, when Runx3 was inactivated and K-Ras was activated by the same targeting method, lung ADCs and other tumors were rapidly induced. In a urethane-induced mouse lung tumor model that recapitulates the features of K-RAS–driven human lung tumors, Runx3 was inactivated in both adenomas (ADs) and ADCs, whereas K-Ras was activated only in ADCs. Together, these results demonstrate that the R-point–associated oncogene surveillance mechanism is abrogated by Runx3 inactivation in AD cells and these cells cannot defend against K-Ras activation, resulting in the transition from AD to ADC. Therefore, K-Ras–activated lung epithelial cells do not evade oncogene surveillance mechanisms; instead, they are selected if they occur in AD cells in which Runx3 has been inactivated.

Targeting Opsin4/Melanopsin with a Novel Small Molecule Suppresses PKC/RAF/MEK/ERK Signaling and Inhibits Lung Adenocarcinoma Progression.

The identification of oncogenic biomolecules as drug targets is an unmet need for the development of clinically effective novel anticancer therapies. In this study, we report for the first time that opsin 4/melanopsin (OPN4) plays a critical role in the pathogenesis of non-small cell lung cancer (NSCLC) and is a potential drug target. Our study has revealed that OPN4 is overexpressed in human lung cancer tissues and cells, and is inversely correlated with patient survival probability. Knocking down expression of OPN4 suppressed cells growth and induced apoptosis in lung cancer cells. We have also found that OPN4, a G protein-coupled receptor, interacted with Gα11 and triggered the PKC/BRAF/MEK/ERKs signaling pathway in lung adenocarcinoma cells. Genetic ablation of OPN4 attenuated the multiplicity and the volume of urethane-induced lung tumors in mice. Importantly, our study provides the first report of AE 51310 (1-[(2,5-dichloro-4-methoxyphenyl)sulfonyl]-3-methylpiperidine) as a small-molecule inhibitor of OPN4, suppressed the anchorage-independent growth of lung cancer cells and the growth of patient-derived xenograft tumors in mice. IMPLICATIONS: Overall, this study unveils the role of OPN4 in NSCLC and suggests that targeting OPN4 with small molecules, such as AE 51310 would be interesting to develop novel anticancer therapies for lung adenocarcinoma.

Dual but not single PD-1 or TIM-3 blockade enhances oncolytic virotherapy in refractory lung cancer

BackgroundProgrammed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) blockade therapy fails in the majority of patients with cancer. Oncolytic viruses represent a new class of therapeutic agents, yet the...

Targeted inhibition of GRP78 by HA15 promotes apoptosis of lung cancer cells accompanied by ER stress and autophagy.

ABSTRACTThis study investigated the pathophysiological role of GRP78 in the survival of lung cancer cells. Lung cancer patient data from public databases were used to analyze the expression of GRP78 and its influence on prognoses. In vivo, GRP78 protein expression was analyzed in an established urethane-induced lung tumor mouse model. In vitro, the effects of targeted inhibition of GRP78 by HA15 in lung cancer cells were assessed, with cell viability analyzed using a CCK-8 assay, cell proliferation using an EdU assay, apoptosis and cell cycle using flow cytometry, subcellular structure using electron microscopy, and relative mRNA and protein expression using RT-PCR, western blotting or immunofluorescence assays. The results showed that GRP78 was highly expressed in the lung tissue of lung cancer mice model or patients, and was associated with a poor prognosis. After inhibition of GRP78 in lung cancer cells by HA15, cell viability was decreased in a dose- and time-dependent manner, proliferation was suppressed and apoptosis promoted. Unfolded protein response signaling pathway proteins were activated, and the autophagy-related proteins and mRNAs were upregulated. Therefore, targeted inhibition of GRP78 by HA15 promotes apoptosis of lung cancer cells accompanied by ER stress and autophagy.

Levamisole in treatment of urethane-induced pulmonary carcinoma in rats

Objective: The potential antitumor effects of the levamisole immunomodulatory agent remain uncertain, and its beneficial effects with increased survival in the adjuvant treatment of malignant tumors are controversial. The present study aims to compare the effects of levamisole with cisplatin in the treatment of urethane-induced lung carcinoma in rats. Methods: Wistar rats were allocated into three groups (n = six each). Group A: mice with lung tumor + treatment with levamisole. Group B: mice with lung tumor + cisplatin treatment. Group C: mice with lung tumor + saline treatment. After 12 weeks of the tumor induction process, the results were validated by ex vivo fluorescence imaging, determining the mean fluorescent intensity in the animal’s lungs. Serum dosages of cytokines and alkaline phosphatase were performed. Results: Mean fluorescence intensity (MFI) in the lungs (ex vivo) was measured in all animals subjected to urethane effects. In levamisole-treated, the intensity (245 +/- 15) was lower than in cisplatin-treated (277 +/- 28), but the difference was not statistically significant (p<0.05). In those treated with saline, the MFI was 680 +/- 57, significantly higher than in the other groups (p<0.05). Dosages of TNF-α (pg/ml), IL-Iβ (pg/ml), IL-6 (pg/ml) and alkaline phosphatase (mg/dl) were significantly lower in levamisole-treated rats than in rats with cisplatin and saline (p<0.005). Conclusion: In conclusion, this study demonstrates the positive influence of levamisole in treatment of urethane-induced lung tumors in rats.

Inhibition of Lung Tumor Development in ApoE Knockout Mice via Enhancement of TREM-1 Dependent NK Cell Cytotoxicity.

Apolipoprotein E (ApoE) is known to regulate lipid homeostasis and associated with atherosclerogenesis. Eventhough atherosclerogenesis is associated with tumor development, the role of ApoE in lung tumorigenesis and metastasis is not clear. Thus, the tumor growth and metastasis were compared in WT and ApoE knockout (KO) mice. Urethane-induced lung tumor incidence and B16F10 lung metastasis in ApoE knockout (KO) mice were significantly reduced in comparison to that in WT mice. Knockdown of ApoE expression in lung cancer cells and B16F10 cells also decreased cancer cell growth and metastasis. The inhibitory effect of ApoE KO on tumor development and metastasis was associated with increase of infiltration of NK cells. NK cells derived from ApoE KO mice showed much greater cytotoxicity than those from WT mice. These cytotoxic effect of NK cells derived from ApoE KO mice was associated with higher expression of Granzyme B, Fas Ligand, IFN-γ, TNF-α, NKG2D, NKp46, and DNAM-1 expression. Triggering receptor expressed on myeloid cell (TREM)-1 is a proinflammatory mediator expressed on NK cells, and is known to be associated with NK cell cytotoxicity. Thus, we investigated the role of TREM-1 on ApoE KO mice originated NK cell mediated cytotoxicity for cancer cells. Blockade of TREM-1 expression with a TREM-1 antagonist prevented NK cell-mediated cytotoxicity. TREM-1 antibody recovered cytotoxic effect of NK cells derived from KO mice of T-bet, which upregulating gene for TREM-1. These data indicate that ApoE KO suppressed lung tumor development and metastasis via increase of TREM-1-dependent anti-tumor activity of NK cells.

Safety and pharmacokinetics of Withaferin-A in advanced stage high grade osteosarcoma: A phase I trial

BackgroundWithaferin-A (WA), an active principle obtained from a traditional Indian herb known as Ashwagandha or the Indian ginseng, has been shown to prevent and cure urethane-induced lung tumors in mice, and also inhibit the growth of transplanted sarcoma in mice. ObjectivesIn this study, we evaluated the safety and pharmacokinetics of WA in patients with advanced stage high-grade osteosarcoma. MethodsA phase I dose escalation study was planned using the classical 3 + 3 design (C33D). Dose escalation cohorts comprised of 72, 108, 144 and 216 mg of WA administered in two to four divided doses per day. Three patients were enrolled in each cohort and the last patient was observed for at least 30 days for any dose-limiting toxicity before progressing to a higher cohort. Pharmacokinetic studies were performed using high performance liquid chromatography (HPLC) technique with sensitivity up to 50 ng/ml. Safety evaluation including clinical examination, detailed history of adverse events, Liver Function Tests , Renal Function Tests and complete blood counts were performed at each visit. WA was administered daily till progression. Common Terminology Criteria for Adverse Events (CTCAE) version 3.0 was used for grading adverse events. ResultsThe formulation used was generally well tolerated. Eleven adverse events of grade 1 or grade 2 severity were observed. No grade 3 or grade 4 adverse events were observed. Elevation of liver enzymes (5/11) and skin rash (2/11) was the most common adverse events. Other adverse effects include fatigue, fever, edema, and diarrhea (one each). None of the patients had detectable levels of WA in circulation. ConclusionThe formulation was well tolerated. However, WA appears to have low oral bioavailability. Further studies with improved formulations are warranted.

RETRACTED: Decreased Lung Tumor Development in SwAPP Mice through the Downregulation of CHI3L1 and STAT 3 Activity via the Upregulation of miRNA342-3p

We previously found that lung tumor development was reduced in a presenilin (PS) Alzheimer’s disease (AD) mouse model. Here, we investigated whether this reducing effect could occur in a different AD mouse model. We investigated urethane-induced (1 mg/g) lung tumor development and melanoma growth in Swedish amyloid precursor protein (SwAPP) transgenic mice. The expression of chitinase-3-like-1 (Chi3L1) increased during lung tumor development and melanoma growth, which was accompanied by an increase in the activity of signal transducer and activator of transcription 3 (STAT3) and the downregulation of miRNA342-3p in wild-type mice. Like tumor development, the expression of Chi3L1 and STAT3 activity was reduced in the SwAPP mice, whereas the expression of miRNA342-3p was upregulated. In addition, Chi3L1 knockdown in the lung cancer and melanoma tissues reduced cancer cell growth and STAT3 activity but enhanced miRNA342-3p expression. However, the miRNA342-3p mimic decreased Chi3L1 expression, cancer cell growth, and STAT3 activity. Moreover, a STAT3 inhibitor reduced Chi3L1 expression and cancer cell growth but enhanced miRNA342-3p expression. These data showed that lung tumor development was reduced through the decrease of Chi3L1 expression via the STAT3-dependent upregulation of miRNA342-3p. This study indicates that lung tumor development could be reduced in SwAPP AD mice.

Preclinical, non-genetic models of lung adenocarcinoma: a comparative survey.

Lung cancer is the leading cause of cancer-related mortality worldwide. Animal models are key in analyzing cancer biology and therapy evaluation.We here compared relevant non-genetic lung cancer models with regard to tumor induction period, incidence, morbidity and mortality rate and the immunological composition of primary tumors and the occurrence of tertiary lymphoid organs (TLO): (I) intraperitoneal Urethane injection (1g/kg), (II) Lewis lung carcinoma (LLC) cell line model (intravenous or subcutaneous), and (III) ex vivo three-dimensional (3D) primary cell culture model established from subcutaneously developed LLC-induced tumors.The incidence of Urethane induced lung tumors was 100% in both, C57BL/6 and BALB/c strains without morbidity or mortality at twenty weeks after injection. The mean size of tumor nodules after Urethane injection was significantly larger in BALB/c mice vs. C57BL/6 (p<0.01). Three times of Urethane injection produced significantly more tumor nodules in both mouse strains compared to one injection (BALB/c: p<0.01; C57BL/6: p<0.05). TLOs were only found in the Urethane-induced model. Although the cell line models also showed 100% induction rate, morbidity was high due to skin ulceration on the inoculation site and the development of pleural effusions in the subcutaneous model and the intravenous model, respectively. Tendencies, but no significant differences (p>0.05) could be found in the count of CD4+, CD8+, F4/80+ and NKp46+ cells in a tumor nodule among investigated models.All discussed models provided a high tumor incidence rate. TLOs were exclusively found in the Urethane-induced model. No significant difference could be found regarding immune cells across models.

Abstract 2987: Identification of new combination therapies for lung tumors harboring KRAS mutations

Abstract Oncogenic mutations in KRAS are frequent in non-small cell lung cancer (NSCLC) and have been associated with poor prognosis and resistance to existing therapies. Thus, new therapeutic strategies are needed to treat these tumors. We have previously shown that NSCLC cells harboring KRAS mutations are more sensitive to inhibition of KRAS downstream effectors MEK and RAF and to treatment with IGF1R inhibitors than their wild-type counterparts. In order to identify complementary targets for the improvement of IGF1R and/or MEK targeting therapies, we performed a whole-genome shRNA screen in KRAS-mutant NSCLC cells. Interestingly, the list of sensitizers to IGF1R inhibitors included several genes encoding components of the mTOR pathway. Viability assays in a panel of lung cancer cell lines confirmed that combinations of IGF1R inhibitors with mTOR inhibitors, both rapalogs and kinase inhibitors, resulted in a synergistic anti-proliferative effect in KRAS-mutant NSCLC cells. Mechanistic investigations demonstrated some differences between the effects of rapalogs and mTOR kinase inhibitors although, in both cases, mTOR inhibitors increased IGF1R and AKT phosphorylation. Combination with IGF1R inhibitors blocked the reactivation of the PI3K pathway resulting in a robust suppression of PI3K and mTORC1 signaling. Notably, the inhibition of these signaling pathways was stronger in KRAS-mutant cells than in wild-type cells. Addition of a MEK inhibitor to the combination produced a more profound and durable suppression of cell proliferation and a stronger induction of apoptosis. Finally, we validated the different drug combinations in mouse models of KRAS-induced NSCLC. Results showed that the three-drug combination produced a marked tumor regression in a NSCLC mouse model driven by mutant Kras and p53 loss-of-function and also in urethane-induced lung tumors. These findings suggest potential novel therapeutic strategies for NSCLC tumors harboring KRAS mutations. Citation Format: Miriam Molina-Arcas, Christopher Moore, Sareena Rana, Febe van Maldegem, Stuart Horswell, David Hancock, Julian Downward. Identification of new combination therapies for lung tumors harboring KRAS mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2987.

Chemopreventive effect of a novel nutrient mixture on lung tumorigenesis induced by urethane in male A/J mice.

Lung cancer, a leading cause of cancer death, is associated with exposure to inhalation carcinogens, most commonly those found in tobacco smoke. We investigated the in vivo effect of dietary supplementation with a nutrient mixture containing lysine, proline, arginine, ascorbic acid, green tea extract, N-acetyl cysteine, selenium, copper and manganese on the development of urethane-induced lung tumors in male A/J mice. After one week of isolation, seven-week-old male A/J mice (n = 25) weighing 17-19 g were randomly divided into three groups: group A (n = 5), group B (n = 10), and group C (n = 10). Mice in groups B and C were each given a single intraperitoneal injection of urethane (1 mg/g body weight) in saline, whereas group A mice received an injection of saline alone. Groups A and B were fed a regular diet, whereas group C was fed the same diet supplemented with 0.5% nutrient mixture. After 20 weeks, mice were sacrificed, lungs were excised and weighed, and tumors were counted and processed for histology. Urethane-challenged mice developed tumors. However, the mean number of tumors and the mean lung weights in the mice on the supplemented diet were significantly reduced, by 49% (P < 0.0001) and 18% (P = 0.0025), respectively, compared to mice on the control diet. We observed neither significant differences in body weight gains nor in diet consumption among the mice. Pulmonary lesions were morphologically similar for both the groups (adenomas), but lesions were smaller in the test group. The results suggest that nutrient mixture has inhibitory potential on the development of mouse lung tumors induced by urethane.

Sex-related differences of urethane and sodium valproate effects on Ki-67 expression in urethane-induced lung tumors of mice.

The aim of the present study was to evaluate sex differences in tumorigenesis by assessing the number of Ki-67-positive cells [Ki-67(+)] in urethane-induced mice lung tumors and the effect of sodium valproate (NaVP) in BALB/c mice. Gonad-intact and gonadectomized female and male mice were divided into the following groups: i) Treated with urethane, ii) treated with urethane and NaVP and iii) gonad-intact or gonadectomized control. Urethane (total 50 mg/mouse) was injected intraperitoneally. The NaVP 0.4% solution was administered orally for 6 months. Histologically, lung tumors were divided into adenomas and adenocarcinomas and assessed immunohistochemically using antibodies against Ki-67. The Ki-67(+) was calculated per one mm2 of a tumor. In adenomas, Ki-67(+) in the urethane-treated gonad-intact males was significantly higher than in females (P=0.001) and in castrated males (P<0.01); Ki-67(+) in adenomas of the urethane-treated gonad-intact males was significantly higher than in urethane-NaVP-treated ones (P<0.04). No significant differences were found in analogous female groups. In adenocarcinomas, Ki-67(+) in urethane-treated gonad-intact males was significantly higher than in females and gonadectomized mice of both sexes (P<0.001), and in ovariectomized females was significantly higher than in ovary-intact group (P=0.01). A significantly higher number of Ki-67(+) cells were observed in gonad-intact adenocarcinomas of the urethane-NaVP-treated females compared with the urethane-treated ones (P<0.001). Comparing between urethane-NaVP-treated gonadectomized males and females in adenocarcinomas, determined that Ki-67(+) was significantly lower in females (P=0.005). In adenocarcinomas, Ki-67(+) in urethane-NaVP-treated gonadectomized males and females was significantly lower than in gonad-intact mice of the same sex (P<0.001). In summary, gonadectomy with NaVP treatment decreased Ki-67(+) in adenocarcinomas for mice of both sexes. The results of the present study indicate sex-related differences in mice lung tumorigenesis, and a sex-related effect of NaVP on progression in urethane-induced BALB/c mice lung tumors.

Immunohistochemical characterization of urethane-induced lung tumors in CB6F1-Tg rasH2 mice.

The purpose of this study was to define the histopathological characteristics of pulmonary neoplastic lesions, especially focusing on the origin of tumor cells, in urethane-treated Tg rasH2 mice. Bronchiolar-alveolar adenomas/adenocarcinomas were observed in the lungs from all of the urethane-treated animals. Immunohistochemically, these tumors showed an alveolar epithelial type II (AE2) cell phenotype demonstrating positive staining of surfactant protein C (SP-C). Cells expressing Clara cell 10 (CC10), a Clara cell marker, were also observed in a scattered manner in some tumors. Several SP-C and CC10 double-positive cells were observed in these tumors. Most of the urethane-induced pulmonary tumors were considered to have an AE2 cell phenotype, but the presence of SP-C and CC10 double positive cells in the tumors of Tg rasH2 mice suggests that some tumors arose from bronchioalveolar stem cells, which are known to express both SP-C and CC10.

P3.01-053 Mouse Models of Primary Lung Cancer - A Thorough Evaluation: Topic: Models of Lung Cancer

Lung cancer is the most prominent cancer in human with the highest mortality rate among cancer patients in both genders nowadays. Several models of primary lung cancer research are in use, however, no systematic evaluation of optimal models is available. Here, we assess and reappraise the most robust models of primary lung cancer for their suitability of cancer evolution and targetability for new therapeutics. Three models of primary lung cancer were evaluated: (I) Carcinogen (urethane or diethylnitrosamine (DEN)) induced lung cancer model, established via three intraperitoneal (i.p.) injections to BALB/c and C57BL/6 mouse strains. Five and ten months after injections, mice were assessed for tumor incidence. Lewis Lung Carcinoma (LLC) cell line was employed for an orthotopic development of lung tumor in syngeneic mouse. The cell line was injected (II) intravenously (i.v.) or (III) subcutaneously (s.c.) to establish lung tumor models in 14 days. Tumor nodules and tumor necrosis were confirmed by microscopy. Immunohistochemistry (IHC) of markers of proliferation (p-Histone3, inhibitor of differentiation 1 (Id1), and Ki67), immune cells (CD4, CD8, B220, F4/80, and NKp46), vascular structure (CD31), stroma (alpha-actin) were performed for a finer characterization of the tumor. Ten months after i.p. injections of carcinogens, we found that the urethane model stably induced tumor nodules (90%: 9/10) when compared to DEN (30%: 3/10). BALB/c strain was significantly more susceptible for the urethane induced tumor development compared to C57BL/6 strain. Injection of LLC cell line via i.v. developed diffuse lung tumor without metastasis to other organs. s.c. injection also stably developed single tumor nodule (∼500mg). IHC revealed that all tumors were consistently positive for the proliferation markers, and F4/80+ cells and CD4+ cells infiltrated into tumors significantly more than CD8+, B220+, or NKp46+ cells. Heterogeneous distributions of CD31+ cells and alpha-actin+ cells were observed in overall tumor models. The urethane-induced lung tumor is reliable and reproducible with a high rate of development and seems superior to DEN induced tumor, but need a long time period to develop. In contrast, the i.v. and s.c. tumor models are established within short time ranges. The tumors developed by s.c. enable for the analysis of the tumor only without adjacent tissue bias. The involvement and characteristics of immune cells found within tumors were comparable across all models. Injections by i.v. or s.c. of cell line to mouse can be considered as an alternative yet convenient model to develop various different types of lung cancers.

Activation of liver X receptor inhibits the development of pulmonary carcinomas induced by 3-methylcholanthrene and butylated hydroxytoluene in BALB/c mice.

We previously reported that LXR ligand, T0901317, inhibited the growth of inoculated Lewis lung carcinoma in C57BL/6 mice by activating IFN-γ production. However, the effects of T0901317 on carcinogen-induced pulmonary carcinomas remain unknown. In this study, we initially conducted a statistical analysis on the data of human lung cancer samples extracted from the TCGA database, and determined that survival rate/time of lung cancer patients and grade of lung adenocarcinoma were positively and negatively related to lung IFN-γ levels, respectively. We then determined the inhibitory effects of T0901317 on mouse pulmonary carcinomas induced by 3-methylcholanthrene (MCA) and butylated hydroxytoluene (BHT) or urethane. We found that T0901317 reduced morbidity and mortality in MCA/BHT-injected BALB/c mice by inhibiting lung adenocarcinoma. T0901317 also protected C57BL/6 mice, but not IFN-γ deficient (IFN-γ−/−, C57BL/6 background) mice, against MCA/BHT-induced lung hyperplasia/inflammation. In addition, we determined that T0901317 inhibited urethane-induced lung tumors in BABL/c mice. Furthermore, we determined that T0901317 prevented metastasis of 4T1 breast cancer cells in BALB/c mice. Administration of T0901317 substantially increased serum IFN-γ levels and lung IFN-γ expression in BABL/c and C57BL/6 mice. Taken together, our study demonstrates that LXR inhibits MCA/BHT-induced pulmonary carcinomas in BABL/c mice and the inhibition is associated with induction of IFN-γ production.

Intranasal Administration of Type V Collagen Reduces Lung Carcinogenesis through Increasing Endothelial and Epithelial Apoptosis in a Urethane-Induced Lung Tumor Model

Type V collagen (Col V) is a "minor" component of normal lung extracellular matrix, which is subjected to decreased and abnormal synthesis in human lung infiltrating adenocarcinoma. We previously reported that a direct link between low amounts of Col V and decreased cell apoptosis may favor cancer cell growth in the mouse lung after chemical carcinogenesis. Moreover, this collagen species was able to trigger DNA fragmentation and impair survival of neoplastic cells. In this study, we have extended our investigation with the aim to obtain further evidence that the death induced by Col V-treatment is of the caspase-9 apoptotic type. We used (1) optical and electron microscopy, (2) quantitation of TUNEL-labeled cells and (3) analysis of the expression levels of Col V and selected genes coding for apoptosis-linked factors, by conventional RT-PCR. BALB/c mice were injected intraperitoneally with 1.5g/kg body weight of urethane. After urethane injection, the animals received intranasal administration of 20µg/20µl of Col V every day during 2months. We report here that Col V treatment was able to determine significant increase in Col V protein and gene expression and in the percentage of TUNEL-positive cells, to up-regulate caspase-9, resulting in low growth of tumor cells. Our data validate chemical carcinogenesis as a suitable "in vivo" model for further and more detailed studies on the molecular mechanisms of the death response induced by Col V in lung infiltrating adenocarcinoma opening new strategies for treatment.

Abstract 2729: Chronic stress promotes lung cancer development via IGF-1R pathway

Abstract Lung cancer is a leading cause of cancer death worldwide. Although smoking is a main cause, approximately 25% of lung cancer cases are not attributable to tobacco use. Epidemiological studies strongly suggest that chronic stress influences cancer development and progression. Recent mechanistic studies showed that biological signaling pathways could contribute to such effects. But the underlying mechanisms for the association between chronic stress and lung cancer development are poorly understood. The purpose of this study is to investigate whether chronic stress can induce lung cancer development and, if so, what is the mechanism underlying the chronic stress-induced lung cancer development. We observed that stress hormone, norepinephrine (NE) increased cell viability in the absence of growth factors, foci formation, and colony formation in human bronchial epithelial cells and activated the type 1 insulin-like growth factor receptor (IGF-1R) pathway. We found that chronic stress resulted in high levels of NE in serum, activation of IGF-1R in lung tissue and promoted urethane-induced lung tumor formation in vivo. Moreover, inhibition of the IGF-1R pathway suppressed NE-induced transformed phenotypes in vitro. We further confirmed that chronic stress-induced lung tumor formation was greater in mice carrying lung-specific human IGF-1R transgene (Tg) than in wild type mice. Our results indicate that chronic stress may promote lung cancer development via the IGF-1R pathway, suggesting that IGF-1R is an effective target for the chemoprevention of stress-induced lung cancer. Citation Format: Hyun-Ji Jang, Hye-Jin Boo, Yujin Jung, Hye-Young Min, Ho-Young Lee. Chronic stress promotes lung cancer development via IGF-1R pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2729. doi:10.1158/1538-7445.AM2015-2729