Murine Lung Adenocarcinoma Cell Line Research Articles

Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma

BackgroundIt has been proposed that anti-angiogenesis therapy could induce tumor "vascular normalization" and further enhance the efficacy of chemotherapy, radiotherapy, target therapy, and immunotherapy for nearly twenty years. However, the detailed molecular mechanism of this phenomenon is still obscure.MethodOverexpression and knockout of CCL28 in human lung adenocarcinoma cell line A549 and murine lung adenocarcinoma cell line LLC, respectively, were utilized to establish mouse models. Single-cell sequencing was performed to analyze the proportion of different cell clusters and metabolic changes in the tumor microenvironment (TME). Immunofluorescence and multiplex immunohistochemistry were conducted in murine tumor tissues and clinical biopsy samples to assess the percentage of pericytes coverage. Primary pericytes were isolated from lung adenocarcinoma tumor tissues using magnetic-activated cell sorting (MACS). These pericytes were then treated with recombinant human CCL28 protein, followed by transwell migration assays and RNA sequencing analysis. Changes in the secretome and metabolome were examined, and verification of retinoic acid metabolism alterations in pericytes was conducted using quantitative real-time PCR, western blotting, and LC–MS technology. Chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) was employed to validate the transcriptional regulatory ability and affinity of RXRα to specific sites at the ANGPT1 promoter.ResultsOur study showed that after undergoing anti-angiogenesis treatment, the tumor exhibited a state of ischemia and hypoxia, leading to an upregulation in the expression of CCL28 in hypoxic lung adenocarcinoma cells by the hypoxia-sensitive transcription factor CEBPB. Increased CCL28 could promote tumor vascular normalization through recruiting and metabolic reprogramming pericytes in the tumor microenvironment. Mechanistically, CCL28 modified the retinoic acid (RA) metabolism and increased ANGPT1 expression via RXRα in pericytes, thereby enhancing the stability of endothelial cells.ConclusionWe reported the details of the molecular mechanisms of "vascular normalization" after anti-angiogenesis therapy for the first time. Our work might provide a prospective molecular marker for guiding the clinical arrangement of combination therapy between anti-angiogenesis treatment and other therapies.

Orthotopic Models Using New, Murine Lung Adenocarcinoma Cell Lines Simulate Human Non-Small Cell Lung Cancer Treated with Immunotherapy.

Understanding tumor-host immune interactions and the mechanisms of lung cancer response to immunotherapy is crucial. Current preclinical models used to study this often fall short of capturing the complexities of human lung cancer and lead to inconclusive results. To bridge the gap, we introduce two new murine monoclonal lung cancer cell lines for use in immunocompetent orthotopic models. We demonstrate how our cell lines exhibit immunohistochemical protein expression (TTF-1, NapA, PD-L1) and common driver mutations (KRAS, p53, and p110α) seen in human lung adenocarcinoma patients, and how our orthotopic models respond to combination immunotherapy in vivo in a way that closely mirrors current clinical outcomes. These new lung adenocarcinoma cell lines provide an invaluable, clinically relevant platform for investigating the intricate dynamics between tumor and the immune system, and thus potentially contributes to a deeper understanding of immunotherapeutic approaches to lung cancer treatment.

Abstract 4372: Epithelial-to-mesenchymal transition-driven secretory program underlies vulnerabilities in lung cancer

Abstract Epithelial-to-mesenchymal transition (EMT) is a driving force behind lung adenocarcinoma (LUAD) progression. Although substantial evidence has been attained in the involvement of EMT and its regulatory mechanisms in cancer, few effective EMT-targeted therapies have been available in the clinic thus far. It is urgent to develop new therapeutic strategies for improving patient outcomes. We have identified RAB6A as a master regulator of EMT-driven secretory trafficking, which coordinates cancer cell invasion and immunosuppression in LUAD. We found that RAB6A expression was associated with a poor prognosis in LUAD patients and that RAB6A inhibition in our LUAD models reversed resistance to immune checkpoint inhibition with PD-L1 blockade, an emerging clinical problem. Thus, RAB6A machinery is a therapeutic target of interest in EMT-driven cancers. Screening approaches to identify selective RAB inhibitors are in their infancy, a challenge that can be overcome only by obtaining insights into the regulation of RAB6A. RAB6A is a small GTPase whose activity is controlled by guanine nucleotide exchange factors (GEFs) like RIC1 and RGP1, and GTPase activating proteins (GAPs) like RABGAP1, as well as other effector proteins. While these GEFs, GAPs, and effectors are all known regulators of RAB6A function and are frequently amplified in different cancer types, their specific functions in cancer progression are still unknown. To establish the causal relationship between these proteins and EMT-driven LUAD progression, we utilize human and murine LUAD cell lines that have been characterized with respect to EMT status. Our findings here show that the intervention of RAB6A activity via depletion of these regulators impairs the recruitment of RAB6A to the Golgi. We demonstrate that knocking down the GEFs, RIC1 and RGP1, significantly decreases invasion while having negligible effects on the migrative and proliferative abilities of these cells. RABGAP1 depletion, on the other hand, reduces cell proliferation but has no effects on the migration and invasion of the cells. Consistent with these results, inactivation of RAB6A abolishes the EMT-dependent pro-metastatic secretion. Mechanistically, the EMT activator, ZEB1, upregulates RGP1 via silencing miR-148a that targets this GEF to activate RAB6A. These findings elucidate the regulatory mechanisms underlying the EMT-driven secretory program, providing a foundation for therapeutic intervention to prevent the progression of LUAD. Citation Format: Kevin L. Fulp, Oluwafunminiyi E. Obaleye, Guan-Yu Xiao. Epithelial-to-mesenchymal transition-driven secretory program underlies vulnerabilities in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4372.

Auranofin Synergizes with the PARP Inhibitor Olaparib to Induce ROS-Mediated Cell Death in Mutant p53 Cancers

Auranofin (AF) is a potent, off-patent thioredoxin reductase (TrxR) inhibitor that efficiently targets cancer via reactive oxygen species (ROS)- and DNA damage-mediated cell death. The goal of this study is to enhance the efficacy of AF as a cancer treatment by combining it with the poly(ADP-ribose) polymerase-1 (PARP) inhibitor olaparib (referred to as 'aurola'). Firstly, we investigated whether mutant p53 can sensitize non-small cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC) cancer cells to AF and olaparib treatment in p53 knock-in and knock-out models with varying p53 protein expression levels. Secondly, we determined the therapeutic range for synergistic cytotoxicity between AF and olaparib and elucidated the underlying molecular cell death mechanisms. Lastly, we evaluated the effectiveness of the combination strategy in a murine 344SQ 3D spheroid and syngeneic in vivo lung cancer model. We demonstrated that high concentrations of AF and olaparib synergistically induced cytotoxicity in NSCLC and PDAC cell lines with low levels of mutant p53 protein that were initially more resistant to AF. The aurola combination also led to the highest accumulation of ROS, which resulted in ROS-dependent cytotoxicity of mutant p53 NSCLC cells through distinct types of cell death, including caspase-3/7-dependent apoptosis, inhibited by Z-VAD-FMK, and lipid peroxidation-dependent ferroptosis, inhibited by ferrostatin-1 and alpha-tocopherol. High concentrations of both compounds were also needed to obtain a synergistic cytotoxic effect in 3D spheroids of the murine lung adenocarcinoma cell line 344SQ, which was interestingly absent in 2D. This cell line was used in a syngeneic mouse model in which the oral administration of aurola significantly delayed the growth of mutant p53 344SQ tumors in 129S2/SvPasCrl mice, while either agent alone had no effect. In addition, RNA sequencing results revealed that AF- and aurola-treated 344SQ tumors were negatively enriched for immune-related gene sets, which is in accordance with AF's anti-inflammatory function as an anti-rheumatic drug. Only 344SQ tumors treated with aurola showed the downregulation of genes related to the cell cycle, potentially explaining the growth inhibitory effect of aurola since no apoptosis-related gene sets were enriched. Overall, this novel combination strategy of oxidative stress induction (AF) with PARP inhibition (olaparib) could be a promising treatment for mutant p53 cancers, although high concentrations of both compounds need to be reached to obtain a substantial cytotoxic effect.

Morphine-3-glucuronide upregulates PD-L1 expression via TLR4 and promotes the immune escape of non-small cell lung cancer.

Objective:Patients with cancer pain are highly dependent on morphine analgesia, but studies have shown a negative correlation between morphine demand and patient outcomes. The long-term use of morphine may result in abnormally elevated serum morphine-3-glucuronide (M3G) levels. Hence, the effects of M3G on tumor progression are worth studying.Methods:The effects of M3G on PD-L1 expressions in human non-small cell lung cancer (NSCLC) cell lines were first evaluated. Activation of TLR4 downstream pathways after M3G treatment was then determined by Western blot. The effects of M3G on human cytotoxic T lymphocytes (CTL) cytotoxicity and INF-γ release was also detected. Finally, the LLC murine lung adenocarcinoma cell line were used to establish a murine lung cancer model, and the effects of M3G on tumor growth and metastasis were determined.Results:M3G promoted the expressions of PD-L1 in the A549 and H1299 cell lines in a TLR4-dependent manner (P < 0.05). M3G activated the PI3K and the NFκB signaling pathways, and this effect was antagonized by a TLR4 pathway inhibitor. A PI3K pathway inhibitor reversed the M3G-mediated PD-L1 upregulation. M3G inhibited the cytotoxicity of CTL on A549 cells and decreased the level of INF-γ. Repeated M3G intraperitoneal injections promoted LLC tumor growth and lung metastasis through the upregulation of tumor expressed PD-L1 and the reduction of CTL in the tumor microenvironment.Conclusions:M3G specifically activated TLR4 in NSCLC cells and upregulated PD-L1 expression through the PI3K signaling pathway, thereby inhibiting CTL cytotoxicity and finally promoting tumor immune escape.

Abstract 621: Tumor suppressor TUSC2 immunogene therapy is synergistic with anti-PD1 in lung cancer syngeneic mouse models

Abstract TUSC2, a pro-apoptotic tumor suppressor gene whose expression is lost or decreased in most lung cancers, activates the innate immune system through initiation of broad spectrum cytokine secretion and natural killer (NK) cell activation. TUSC2 delivered systemically by nanovesicles has mediated tumor regression in metastatic non-small cell lung cancer clinical trials. We studied the effect of TUSC2 on immune cell populations and the anti-tumor activity of TUSC2 in combination with anti-PD1 checkpoint blockade in two syngeneic mouse models: C57BL/6 mice subcutaneously injected with murine lung adenocarcinoma cell line CMT/167-luc cells (KrasG12V mutation) and 344SQ (KrasG12D allele and a knock-in Trp53R172HΔG allele) adenocarcinomas which metastasize to the lung in 129S2 mice. Tumor growth was monitored by scoring ex-vivo luminescence using the IVIS Imaging System 200. Multi-color flow cytometry was used for immune profiling of circulating immune cells after nanovesicle mediated TUSC2 intravenous injection. Cytokine gene expression in response to TUSC2 in sorted immune subpopulations was determined by real-time PCR. Tumor growth was significantly reduced with TUSC2 treatment compared with no treatment in both subcutaneous and metastatic mouse models. Synergistic anti-tumor activity was observed when TUSC2 was combined with anti-PD1 verified in five independent experiments. In the lung metastasis model, mice treated with TUSC2 + anti-PD1 lived significantly longer than with single agent treatment. Circulating NK cells increased three fold following TUSC2 nanovesicle intravenous injection both in tumor free and tumor bearing mice which correlated with tumor regression and survival. Cytotoxic T lymphocyte responses were increased whereas Tregs and MDSCs decreased with TUSC2 alone and TUSC2+anti-PD1 treatment. The levels of T cell checkpoint markers PD1, CTLA-4, LAG-3, and TIM-3 evaluated by flow cytometry were decreased after TUSC2 treatment. TUSC2 anti-tumor response was abolished when NK cells were depleted indicating NK cells are important mediators of the TUSC2 treatment effect. Single cell suspension analysis by flow cytometry showed high numbers of NK cells infiltrating lung tumor metastases after TUSC2 treatment. The number of tumor nodules in the lung was significantly less following treatment with TUSC2 nanovesicles compared with control. IL-15 gene expression which mediates NK cell proliferation, was increased by TUSC2. In conclusion, systemic TUSC2 nanovesicle immunogene therapy combined with checkpoint blockade showed synergistic anti-tumor efficacy and activated the immune system through upregulation of NK cells and CTL and downregulation of regulatory cells. Citation Format: Ismail M. Meraz, Mourad Majidi, RuPing Shao, Meng Feng, Xiaobo Cao, David Rice, Boris Sepesi, Lin Ji, Jack Roth. Tumor suppressor TUSC2 immunogene therapy is synergistic with anti-PD1 in lung cancer syngeneic mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 621. doi:10.1158/1538-7445.AM2017-621

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract B50: A novel immunocompetent murine model for replicating oncolytic adenoviral therapy

Abstract Oncolytic adenoviruses are able to replicate selectively in cancerous but not normal human cells and are under investigation as potential cancer therapeutics. Unfortunately, mice are poor model systems for therapy with replication competent adenoviruses because murine cells are generally unable to produce infectious viral progeny. In order to model oncolytic adenovirus treatment, researchers have used human tumor xenografts that support viral replication in immunodeficient mice. Because xenografts require an immunodeficient host, xenograft model systems do not reflect a host's adaptive immune response against the virus and cannot model the potential for an immunostimulatory virus to induce an anti-tumor immune response. Using the tumor-selective oncolytic adenovirus TAV-255 previously developed in our laboratory, we find that the murine lung adenocarcinoma cell line LKR-13 supports adenoviral infection and generates infectious viral progeny. Western blotting showed that LKR-13 cells express the coxsackie and adenovirus receptor (CAR) to allow viral infection, and that infected cells express the viral protein E1A. MTT assays, crystal violet staining, and clonogenic assays showed that TAV-255 kills LKR-13 cells in a dose-dependent and time-dependent manner with similar potency and latency of infection as a panel of human cancer cell lines, with more than 50% cell death of murine LKR-13 cells and various human cancer cells (T1, 882 and A549) within 6 days of infection at a multiplicity of infection of 10. It produces infectious progeny from LKR-13 cells at titres similar to other human cancer cell lines. Other tested murine cancer cell lines do not share these features; for example, the CT-26 colon cancer cell line does not express CAR and is resistant TAV-255 infection, while the murine sarcoma cell line F244 is readily infected by adenovirus and produces the viral protein E1A but does not produce infectious progeny. Based on these in-vitro findings, we investigated LKR-13 cells as an in-vivo model system for replicating oncolytic adenoviruses. The LKR-13 cell line is derived from 129/Sv mice, and using fully immunocompetent 129/s4 mice, subcutaneous injection of 5x106 LKR-13 cells led to tumor formation in all injected mice. Intratumoral injection of 3x107 plaque forming units of TAV-255 in established tumors (greater than 200 mm3) caused a significant reduction in tumor growth: Eight days after treatment, tumors in mice treated with TAV-255 were 339 mm3 +/- 26 mm3 (n=3), and tumors in mice treated with PBS were 578 mm3 +/- 49 mm3 (n=5) (p=0.013). This model system represents the first fully immunocompetent mouse model for cancer treatment with replicating oncolytic adenoviruses, and will be useful to study the therapeutic effect of oncolytic adenoviruses in general and particularly immunostimulatory viruses designed to evoke an anti-tumor immune response. Citation Format: Lingzhi Zhang, Farah Hedjran, Christopher Larson, Min Yan, Tony Reid. A novel immunocompetent murine model for replicating oncolytic adenoviral therapy. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B50.

Effect of DTPP-mediated photodynamic therapy on cell morphology, viability, cell cycle, and cytotoxicity in a murine lung adenocarcinoma cell line.

Photodynamic therapy (PDT) involves the administration and activation of photosensitizing reagents in cancer tissues to induce cytotoxicity. Here we examined the effects of 5-5- (4-N, N-diacetoxylphenyl)-10,15,20- tetraphenylporphyrin (DTPP) -mediated PDT on cell morphology, viability, cell cycle, and cytotoxicity in a murine lung adenocarcinoma cell line. LA795 murine lung adenocarcinoma cell line was used in the study, with cellular uptake of DTPP being quantified by a UV-visible spectrophotometer. The subcellular localization of DTPP was detected by confocal laser scanning microscopy, alteration of cell morphology after PDT was observed by an inverted light microscope, and late-stage apoptosis was examined by terminal dUTP nick end labeling (TUNEL) . The effects of influencing factors on cytotoxicity of PDT in LA795 cells was investigated with varying concentrations of DTPP, energy densities, power densities, and antioxidants by 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Effects of PDT on cell cycle and plasma membrane integrity were studied by flow cytometry analysis. The uptake of DTPP by LA795 cells reached maximum after incubation for 24 h. Confocal laser scanning microscopy showed that DTPP was mainly in the mitochondrion, and slight localization was detected in the lysosomes. Cellular inhibitory effects increased with increased irradiation dose and DTPP concentration, while unactivated DTPP had low toxicity. Flow cytometry analysis revealed that DTPP-PDT-treated cells showed S phase arrest. Cell membrane damage initiation, repair, and irreversible damage were observed at 2, 4, and 5 h after DTPP-PDT , respectively. Together, our results demonstrated cell apoptosis, compromised viability, and cell cycle S phase arrest of LA795 in response to DTPP-PDT , while no effect on the lung cancer cells was observed with irradiation or photosensitizer treatment alone.

TGF-beta specifically enhances the metastatic attributes of murine lung adenocarcinoma: implications for human non-small cell lung cancer

Lung cancer is the most frequent and one of the most deadly cancer types and is classified into small cell lung cancer and non-small cell lung cancer (NSCLC). Transforming growth factor beta (TGFβ) regulates a wide array of cell functions and plays a major role in lung diseases, including NSCLC. TGFβ signals through the complex of TGFβ type I and type II receptors, triggering Smad and non-Smad signaling pathways such as PI3K/Akt and MEK1/ERK. We investigated the role of TGFβ1 on the progression of the murine lung adenocarcinoma cell line LP07. Furthermore, we undertook a retrospective study with tissue samples from stage I and II NSCLC patients to assess the clinical pathologic role and prognostic significance of TβRI expression. We demonstrated that although lung cancer cell monolayers responded to TGFβ1 anti-mitogenic effects and TGFβ1 pulse (24 h treatment) delayed tumor growth at primary site; a switch towards malignant progression upon TGFβ1 treatment was observed at the metastatic site. In our model, TGFβ1 modulated in vitro clonogenicity, protected against stress-induced apoptosis and increased adhesion, spreading, lung retention and metastatic outgrowth. PI3K and MEK1 signaling pathways were involved in TGFβ1-mediated metastasis stimulation. Several of these TGFβ responses were also observed in human NSCLC cell lines. In addition, we found that a higher expression of TβRI in human lung tumors is associated with poor patient's overall survival by univariate analysis, while multivariate analysis did not reach statistical significance. Although additional detailed analysis of the endogenous signaling in vivo and in vitro is needed, these studies may provide novel molecular targets for the treatment of lung cancer.

Abstract A59: Dixdc1 links the LKB1 tumor suppressor to Snail, noncanonical Wnt signaling, and metastasis

Abstract The kinase LKB1 is a tumor suppressor mutated in familial and sporadic human cancers, whose loss is uniquely coupled with metastatic progression. Although LKB1 controls growth and metabolism through phosphorylation of AMPK, it also phosphorylates the related MARK/ Par1 kinases, which play conserved roles in cell polarity and cytoskeletal dynamics. In a screen for novel targets of LKB1-dependent signals, we identified the scaffold protein DIXDC1 as a substrate of MARK1 which controls cell migration and invasion. DIXDC1 localizes to focal adhesions, and like LKB1, its depletion results in hyperactivation of focal adhesion kinase and subsequent increases in the EMT-transcription factors Snail and Slug. Depletion of LKB1 or DIXDC1 results in Snail-dependent induction of the noncanonical Wnt5a and Wnt5b, which are required for the increased invasive behavior of these cells. Studies on a panel of murine lung adenocarcinoma cell lines from defined stages of metastatic progression reveals that DIXDC1 depletion was sufficient to promote metastasis, while Wnt5a and Wnt5b are required for metastatic behavior in these cells. DIXDC1 is frequently downregulated in human cancers, and inversely correlates with Snail and Wnt5 family members expression. This study reveals the first downstream effector of LKB1 in tumor suppression that is independent of AMPK and identifies a novel signaling pathway that provides a molecular explanation for how LKB1 controls EMT and metastasis. Citation Format: Jonathan M. Goodwin, Robert U. Svensson, David B. Shackelford, Monte M. Winslow, Reuben J. Shaw. Dixdc1 links the LKB1 tumor suppressor to Snail, noncanonical Wnt signaling, and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A59.

Abstract 5236: Potential loss of p53/p21 function in a murine lung adenocarcinoma cell line prevents TGFβ1-induced dormancy despite p38α/β activation and DEC2 and p27 induction

Abstract Disseminated tumor cells can reside in target organs in a state of dormancy (growth arrest) for prolonged periods before developing into overt lesions. Studies using the HEp3 model of head and neck squamous cell carcinoma (HNSCC) showed that a low ERK:p38 signaling ratio predicts for cellular dormancy while the opposite prompts active proliferation. Further, p38 activation induces the expression of p53 and the tumor/metastasis suppressor BHLHB3/DEC2, while inhibiting c-jun and FoxM1 expression. Also, at least p53 and DEC2 are positively regulated by transforming growth factor beta (TGFβ). Preliminary data studying the murine lung adenocarcinoma cell line LP07 suggested that TGFβ1 can only delay primary tumor growth with no effect on spontaneous metastasis development. These studies motivated us to test whether the effects of TGFβ1 signaling in the LP07 model might be short-circuited to prevent the activation of a tumor cell dormancy program as that described in HNSCC. Flow cytometry analysis showed that TGFβ1 treatment mediated arrest in G0/G1 phase of the cell cycle in LP07 cells, which was consistent with p27 upregulation observed by Western blot. Concomitantly, TGFβ1 activated the transcriptions factors Smad2/3 and inhibited proliferation, all these events blocked by an inhibitor of TGFβ-receptor serine kinase activity. Consistent with these data, LP07 cells exhibited a high ERK:p38 ratio that was lowered by TGFβ1, primarily due to a strong p38 activation. Accordingly, we observed in vivo growth inhibition of TGFβ1-pretreated cells upon subcutaneous implantation in syngeneic BALB/c mice. However, sustained in vivo growth arrest was not observed. We also found by qPCR an increased level of DEC2 mRNA in TGFβ1-treated cells. LP07 cells overexpressing DEC2 or a constitutive active mutant of p38 had increased p27 expression as assessed by Western blot, suggesting that the p38-&amp;gt;DEC2 pathway can be re-activated. Upon treatment of the cells with a MEK1/2 inhibitor, p27 was also upregulated and phospho-histone H3 was downregulated. p53 and p21 were refractory to modulation by these treatments, even following overexpression of wild type p53. This suggests that p53 function might be disrupted and/or that p21 may be epigenetically silenced in this cell line. In conclusion, we have found that the metastatic LP07 cell line displays an ERK/p38 ratio and downstream signaling that predict for an efficient escape from dormancy. While the p38-&amp;gt;DEC2-p27 pathway could be restored by establishing a low ERK/p38 signaling ratio, absence of functional p53 and p21 induction most likely explains why TGFβ1 only attenuates tumor growth. If p53 and p21 (also a target of DEC2) are silenced by DNA promoter methylation it is possible that restoration of p21 promoter sensitivity to DEC2 using 5-Aza-C may allow for reprogramming LP07 cells into a prolonged dormant state. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5236. doi:1538-7445.AM2012-5236

Response and Resistance to NF-κB Inhibitors in Mouse Models of Lung Adenocarcinoma

Lung adenocarcinoma is a leading cause of cancer death worldwide. We recently showed that genetic inhibition of the NF-κB pathway affects both the initiation and the maintenance of lung cancer, identifying this pathway as a promising therapeutic target. In this investigation, we tested the efficacy of small-molecule NF-κB inhibitors in mouse models of lung cancer. In murine lung adenocarcinoma cell lines with high NF-κB activity, the proteasome inhibitor bortezomib efficiently reduced nuclear p65, repressed NF-κB target genes, and rapidly induced apoptosis. Bortezomib also induced lung tumor regression and prolonged survival in tumor-bearing Kras(LSL-G12D/wt);p53(flox/flox) mice but not in Kras(LSL-G12D/wt) mice. After repeated treatment, initially sensitive lung tumors became resistant to bortezomib. A second NF-κB inhibitor, Bay-117082, showed similar therapeutic efficacy and acquired resistance in mice. Our results using preclinical mouse models support the NF-κB pathway as a potential therapeutic target for a defined subset of lung adenocarcinoma. Using small-molecule compounds that inhibit NF-κB activity, we provide evidence that NF-κB inhibition has therapeutic efficacy in the treatment of lung cancer. Our results also illustrate the value of mouse models in validating new drug targets in vivo and indicate that acquired chemoresistance may later influence bortezomib treatment in lung cancer.

Abstract 400: Modulation of the metastatic microenvironment by soluble factors released by lung adenocarcinoma cells

Abstract Soluble factors from primary tumors can modify target organs for metastasis through mobilization of bone marrow derived cells (BMDCs), which are recruited to tumors and to metastatic target organs and facilitate tumor growth and metastasis. Here we aimed to study early cellular and molecular events occurring in the lung, the metastatic target organ of the murine lung adenocarcinoma cell line LP07. We assessed by flow cytometry the numbers of BMDC positive for VEGFR1, SCF, CD34 or CXCR4 and the numbers of myeloid suppressor cells (CD11b+/Gr1+) and regulatory T cells (CD4+/CD25+/FOXP3+) in single cell lung suspensions obtained from 15-day tumor-bearing and control mice. Control and tumor-conditioned lungs of inoculated mice showed comparable amounts of these cell populations. To assess whether the primary tumor stimulates subsequent formation of macroscopic metastasis, mice bearing an intraperitoneal millipore chamber -that allows the diffusion of soluble molecules but not cell extravasation- loaded with LP07 cells or medium, were challenged with a LP07 intravenous inoculum at day 15. The presence of LP07 loaded chambers decreased the number of lung metastases three weeks after the inoculum compared to control mice (p≪0.05). In a different approach, mice were daily injected for 15 days with LP07 conditioned medium or control medium, and then challenged with a LP07 intravenous inoculum. When evaluated at day 21 post LP07 cell inoculation, the number of metastatic lung nodules was significantly lower (p≪0.05) in the group treated with LP07 conditioned medium. Besides, when we assessed matrix metalloproteinase (MMP) activity in lung homogenates from 15 day-tumor bearing and control mice, we found a significant increase in MMP-9 activity (p≪0.05) as assessed by semi-quantitative zymography in lungs from tumor-bearing mice.In conclusion, in the LP07 lung adenocarcinoma model, tumor cells secrete soluble factors that induce certain changes in the metastatic target organ, as the increase of MMP-9 activity. Moreover, in this experimental metastatic approach, a decrease in metastasis development was observed; the mechanisms underlying this phenomenon deserve further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 400. doi:10.1158/1538-7445.AM2011-400

Zoledronic acid induces cell-cycle prolongation in murine lung cancer cells by perturbing cyclin and Ras expression

Zoledronic acid (ZOL) was shown earlier to prolong survival in animal models of lung cancer. The aim of this study was to examine whether alteration of intracellular cyclins, cyclin-dependent kinases, cyclin-dependent kinase inhibitors, retinoblastoma, and Ras protein expression and E2F localization are among the possible antilung cancer mechanisms driven by ZOL. Furthermore, we used geranylgeraniol to test whether the mevalonate pathway is involved in the antitumor effects of ZOL against lung cancer. Line-1 cells, a murine lung adenocarcinoma cell line, were examined. ZOL significantly slowed the growth of these cells both in vitro and in vivo. The ZOL-treated cells typically arrested at the S/G2/M phase of the cell cycle, accompanied by increased intracellular levels of cyclin A, B1, and CDC2 and decreased levels of cyclin D, p21, p27, phosphorylated retinoblastoma, and Ras. In addition, ZOL affected the distribution of E2F. When geranylgeraniol was added to the ZOL-treated cells, either in vitro or in vivo, tumor growth, cell-cycle progression, the expression of certain cyclins, and cyclin-related regulatory proteins were partially returned to that of untreated controls. Therefore, ZOL elicits cell-cycle prolongation that seems to be associated with alterations in the levels of certain cyclins and cyclin-related regulatory proteins. Furthermore, the mevalonate pathway regulates ZOL-induced murine lung cancer inhibition both in vitro and in vivo.

Abstract LB-151: Targeted gene resequencing in a K-ras-dependent mouse model of metastatic lung adenocarcinoma

Abstract Progression of lung adenocarcinoma to invasive, metastatic disease is responsible for the majority of deaths from lung cancer and remains incompletely understood. We have developed a genetically-engineered mouse model of lung adenocarcinoma initiated by mutant K-ras and loss of p53 that develops invasive, metastatic disease. In order to identify the genes and pathways that cooperate with oncogenic K-ras and loss of p53 to contribute to tumor development, progression, and metastasis, we have utilized in situ exon capture and massively parallel DNA sequencing to identify somatically-acquired point mutations in advanced stage murine lung adenocarcinoma cell lines. We have initially chosen a target set of 1283 cancer-relevant genes to develop our capture, sequencing, and bioinformatics platforms. Using tumor cell lines derived from K-ras-dependent tumors, we have successfully developed and refined these methods and have identified somatic mutations in our test samples. These mutations have been validated by conventional methods in the tumor cell line and founder tumor DNA. We have developed a production phase target gene set consisting of 1909 cancer-relevant genes and have initiated capture and sequencing of additional murine lung adenocarcinoma cell lines and tumors. We believe these datasets will expand the understanding of the biology of tumor progression and serve as an important biological filter for human cancer gene re-sequencing efforts. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-151.

The Kinetics and Redox State of Nitric Oxide Determine the Biological Consequences in Lung Adenocarcinoma

Few studies have explored the mechanistic basis for the apparent paradoxical effects of nitric oxide and its interrelated redox species (NO_X) in cancer biology. Our aim was to determine the differential effects of the redox state and kinetics of nitrosative species on the key cancer processes of apoptosis. Therefore, a murine lung adenocarcinoma cell line was exposed to various NO_X donor compounds differing in redox state and delivery kinetics. DNA strand breaks (DSBs) were measured by the alkaline single-cell gel electrophoresis assay (the COMET assay) and correlated with cell viability by the MTT and soft agar colony assays, while caspase enzymatic activity was measured using an in vitro fluorogenic caspase assay. Finally, cDNA microarrays defined apoptosis-related gene expression alterations resultant from these NO_X donors. Exogenous NO_X differentially influences DSBs, and apoptosis-related cell death and expression based on the redox state and kinetics of NO_X delivery. In our murine lung adenocarcinoma model we have demonstrated differential effects of NO_X based on the mode of delivery and redox state. These data suggest that the development of NO_X-based cancer chemotherapy must consider the redox state and kinetics of delivery into their logical design.

Zoledronic acid is unable to induce apoptosis, but slows tumor growth and prolongs survival for non-small-cell lung cancers

Although zoledronic acid (ZOL), a third-generation nitrogen-containing bisphosphonate, has been identified as an attractive therapeutic agent against breast cancer, prostate cancer, multiple myeloma as well as small-cell lung cancer (SCLC), as best as we are aware, the anti-tumor effect of ZOL upon non-small-cell lung cancer (NSCLC) remains to be effectively investigated. This study examined the effects of ZOL upon the line-1 tumor cell, using a murine lung adenocarcinoma cell line similar to the behavior of human lung adenocarcinoma. We investigated the anti-tumor effects of ZOL (3-100 microM) on line-1 tumor cells in vitro, including cellular proliferation, by means of an MTT assay, cell-cycle analysis by flow cytometry and by assessing the level of apoptosis by annexin V/propidium iodide (PI) and 4'-6-diamidino-2-phenylindole (DAPI) staining. Further, we evaluated the growth and survival of line-1 tumor cells following ZOL treatment (1 microg/kg/week) using an animal model. We also examined the in vivo cell-cycle pattern using lacZ-expressing line-1 cells (line-1/lacZ). ZOL significantly slowed the line-1 tumor growth in a dose-dependent manner in vitro. The treated line-1 tumor cells typically arrested at the S/G2/M-phase of the cell-cycle following ZOL exposure, but no apoptotic cells could be detected by either annexin V/PI or DAPI staining. When the ZOL was washed out, the drug-inhibited cells continued to proliferate again and the cell-cycle prolongation elicited earlier by the drug, then disappeared. Within 72-96 h following drug removal, the cell-cycle of the treated cells revealed a similar distribution to that of the untreated controls. In vivo studies demonstrated that ZOL significantly slowed the line-1 tumor growth. Indeed, mice lived significantly longer when they had been ZOL-treated than was the case for untreated mice (p<0.05). Using line-1/lacZ cells, the in vivo cell-cycle distribution of line-1 tumor cells subsequent to ZOL exposure revealed S/G2/M-phase arrest that was identical to the in vitro culture. ZOL maintains the potential to reduce tumor burden and prolong survival for murine pulmonary adenocarcinoma. The flow cytometrical analysis of cell-cycle demonstrated that ZOL induces no apoptosis but is able to arrest line-1 tumor cells at the S/G2/M-phase. Although the clinical relevance of these results warrants verification for human lung cancer patients, ZOL combined with chemotherapy and/or radiotherapy appears to be a new therapeutic strategy for the effective treatment of NSCLC.

Invasion of spheroid of murine lung adenocarcinoma (LA795) cells into embryonic chick heart fragments

Anin vitro spheriod invasion model of tumor cell was established by using murine lung adenocarcinoma cell line (LA795) and precultured embryonic chick heart fragment (PHF). The spheroid of LA795 cells were prepared by incubating a suspension of trypsinized LA795 cells on a gyratory shaker. Spheroid aggregates of LA795 cells in diameter of 0.2 mm were selected and confronted with PHF (diameter of 0.4 mm) on semi-solid medium for 3 – 4 hours, then, individual confronting pairs were transferred into fluid medium for further co-culture on gyratory shaker. After 1,3,5 and 7 days, multiple confronting pairs were processed for histological and ultrastructural study. The invasive capacity and the invasion process of LA795 cells were examined and observed. The results demonstrated that LA795 cell line has a high capacity of invasion and high malignancyin vitro. This spheroid invasion model is very useful for studying mechanism of invasiveness of tumor cellsin, vitro.