Mouse Lung Tumor Cells Research Articles

Bmi1 Resistance Pathway and Immune Checkpoint Blockade in Lung Cancer

Combined chemotherapy and radiation is a standard therapy for locally advanced lung cancers. However, the resistance mechanisms to these therapies and how they affect further adjuvant immunotherapy are not well understood. Our previous studies have shown that radiation led to the activation of Bmi1 as a mechanism of radiation resistance, which in turn upregulated the inhibitory immune checkpoint ligand PD-L1. Our hypothesis is that inhibition of Bmi1, a target for chemoradiotherapy resistance, with concurrent PD-L1 checkpoint blockade would have a synergistic enhanced immune response after chemoradiation therapy in a preclinical model of lung cancer.A small molecule inhibitor, PTC-596 (PTC Therapeutics, NJ), has been shown to inhibit the expression of Bmi1. For in vitro studies, we tested the effects of PTC-596 on the expression of both Bmi1 and PD-L1 in several lung tumor cell lines. To create a mouse model that simulates the PACIFIC trial treatment regimen, Lewis lung carcinoma cells (LLC1; 1 x 106 unsorted cells in 100 ul) derived in C57BL/6J strain were injected subcutaneously into the flank of immunocompetent C67BL/6J mice. When average tumor volume reached 100 mm3, the mice were treated with phase 1 treatment of radiation (dose 30 Gy in 10 fractions daily) delivered through SAARP platform and cisplatin (2mg/kg, every 3rd day for 10 days) and then randomized to receive phase 2 treatments: 1). Vehicle control every day; 2). Inhibitory anti-PD-L1 antibody every other day (200mg/kg, every 3 days for 4 cycles); 3). PTC-596 once a day (12.5 mg/kg, once a day); 4). Combined inhibitory anti-PD-1 antibody and PTC-596. Each treatment group had 10 mice.In both human and mouse lung tumor cell lines, fractionated radiation treatments upregulated Bmi1 expression and also increase the frequency of Bmi1-high, PD-L1-high expressing cells. Treatment with PTC-596 downregulated expression of Bmi1 and PD-L1 in these cells. In the PACIFIC study mouse model, cisplatin chemotherapy with fractionated radiation resulted significant growth delay than compared to no treatment. In the phase 2 of adjuvant treatment, the anti-PD-L1 antibody plus PTC-596 combination group, but not in groups receiving either treatment alone, had continued significant tumor size reduction. Immunological markers are being analyzed to determine if the immune response is enhanced.These results suggest that chemoradiation therapy leads to the activation of Bmi1 as a resistance mechanism, which in turn results in the activation of inhibitory immune checkpoint PD-L1 leading to immune escape and acquired resistance to immunotherapy. Treatment with PTC-596 demonstrated synergistic benefits to anti-PD-L1 immunotherapy after pre-chemoradiation treatment of preclinical mouse lung tumor model.

AMOTL2 inhibits JUN Thr239 dephosphorylation by binding PPP2R2A to suppress the proliferation in non-small cell lung cancer cells

Protein phosphatase 2A (PP2A) complex comprises an extended family of intracellular protein serine/threonine phosphatases, that participate in different signaling transduction pathways. Different functions of PP2As are determined by the variety of regulatory subunits. In this study, CRISPR/Cas9-mediated loss-of-function screen revealed that PPP2R2A downregulation suppressed cell growth in NSCLC cells. AMOTL2 was identified and confirmed as a novel binding partner of PPP2R2A in NSCLC cells by mass spectrometry, CO-IP, GST pull-down and immunofluorescence. Upregulation of AMOTL2 also led to cell proliferation delay in human and mouse lung tumor cells. The proto-oncogene JUN is a key subunit of activator protein-1 (AP-1) transcription factor which plays crucial role in regulating tumorigenesis and its activity is negatively regulated by the phosphorylation at T239. Our results showed that either AMOTL2 upregulation or PPP2R2A downregulation led to great increase in JUN T239 phosphorylation. AMOTL2 bound PPP2R2A in cytoplasm, which reduced nuclear localization of PPP2R2A. In conclusion, AMOTL2 and PPP2R2A act respectively as negative and positive regulator of cell growth in NSCLC cells and function in the AMOTL2-PPP2R2A-JUN axis, in which AMOTL2 inhibits the entry of PPP2R2A into the nucleus to dephosphorylate JUN at T239.

PTGES/PGE2 signaling links immunosuppression and lung metastasis in Gprc5a-knockout mouse model

Chronic inflammation has been linked to promotion of tumorigenesis and metastasis in lung. However, due to lack of a relevant animal model for characterization, the underlying mechanism remains elusive. Lung tumor suppressor gene Gprc5a-knockout (ko) mice are susceptible to lung inflammation, tumorigenesis and metastasis, which resembles the pathological features in human patients. Here, we showed that PTGES/PGE2 signaling was highly associated with lung tumorigenesis and metastasis in Gprc5a-ko mice. Interestingly, Ptges-knockout in mouse lung tumor cells, although reduced their stemness and EMT-like features, still formed tumors and lung metastasis in immune-deficient nude mice, but not in immune-competent mice. This suggests that the major role of PTGES/PGE2 signaling in tumorigenicity and lung metastasis is through immunosuppression. Mechanistically, PTGES/PGE2 signaling intrinsically endows tumor cells resistant to T-cell cytotoxicity, and induces cytokines extrinsically for MDSC recruitment, which is crucial for suppression of T-cell immunity. Importantly, targeting PGE2 signaling in Gprc5a-ko mice by PTGES inhibitor suppressed MDSC recruitment, restored T cells, and significantly repressed lung metastasis. Thus, PTGES/PGE2 signaling links immunosuppression and metastasis in an inflammatory lung microenvironment of Gprc5a-ko mouse model.

Enhanced gene delivery in tumor cells using chemical carriers and mechanical loadings.

Intracellular delivery of DNA is considered a challenge in biological research and treatment of diseases. The previously reported transfection rate by commercially available transfection reagents in cancer cell lines, such as the mouse lung tumor cell line (TC-1), is very low. The purpose of this study is to introduce and optimize an efficient gene transfection method by mechanical approaches. The combinatory transfection effect of mechanical treatments and conventional chemical carriers is also investigated on a formerly reported hard-to-transfect cell line (TC-1). To study the effect of mechanical loadings on transfection rate, TC-1 tumor cells are subjected to uniaxial cyclic stretch, equiaxial cyclic stretch, and shear stress. The TurboFect transfection reagent is exerted for chemical transfection purposes. The pEGFP-N1 vector encoding the green fluorescent protein (GFP) expression is utilized to determine gene delivery into the cells. The results show a significant DNA delivery rate (by ~30%) in mechanically transfected cells compared to the samples that were transfected with chemical carriers. Moreover, the simultaneous treatment of TC-1 tumor cells with chemical carriers and mechanical loadings significantly increases the gene transfection rate up to ~ 63% after 24 h post-transfection. Our results suggest that the simultaneous use of mechanical loading and chemical reagent can be a promising approach in delivering cargoes into cells with low transfection potentials and lead to efficient cancer treatments.

Functional characterization of RAD52 as a lung cancer susceptibility gene in the 12p13.33 locus.

Recent genome-wide association studies have identified variations in the recombination repair gene, RAD52, that are associated with increased lung cancer risk, and particularly with the development of lung squamous cell carcinomas (LUSC). As LUSC development is strongly associated with smoking, DNA repair is increased in the lung tissues of smokers, presumably because of ongoing DNA damage from exposure to tobacco smoke. A key player in the DNA damage response, RAD52 plays a role in DNA strand exchange and annealing during homologous recombination (HR) in mammalian cells. In this study, we discovered two cis-expression quantitative trait loci (eQTL) SNPs in the RAD52 gene that are associated with its expression and are also associated with LUSC risk. In addition, we report that amplification of the genomic region 12p13.33, which contains the RAD52 gene, is significantly associated with the development of LUSC in the TCGA database and that somatic overexpression of RAD52 was confirmed to be significant in LUSC tumors from our own patient cohort. Consistent with these genetic findings, we demonstrate that blockade of Rad52 slows cell growth and induces senescence in mouse bronchial epithelial cells. In contrast, overexpression of Rad52 leads to an increased rate of cell proliferation. We show that depletion of Rad52 in mouse lung tumor cells alters cell cycle distribution and increases DNA damage accumulation associated with increased tumor cell death. Our genetic and functional data implicate RAD52 as a significant determinant of risk in the development of LUSC.

Knockdown of NF-κB p65 subunit expression suppresses growth of nude mouse lung tumor cell xenografts by activation of Bax apoptotic pathway.

Nuclear factor-kappaB (NF-κB) is an important transcriptional factor and regulates avariety of pathophysiologic process involved in cell survival and death. The present study assesses the effects of NF-κB p65 subunit knockdown in suppression of nude mouse lung tumor cell xenografts and understands the underlying molecular events.A nude mouse Lewis lung carcinoma cell xenograft model was established and the mice were intraperitoneally injected with NF-κB p65 siRNA and sacrificed after two weeks of tumor cell injection. Tumor xenografts were harvested for TUNEL, Western blot, and qRT-PCR analyses.Compared to the PBS-treated or the negative control (NC) siRNA-treated mice, tumor xenograft weight and volume was significantly decreased in the NF-κB p65 siRNA-treated mice. The TUNEL positive (apoptosis) cells in xenograft sections were 45 ± 5 in PBS and 38 ± 3 in NC siRNA, but increased to 271 ± 11 in p65 siRNA-treated mice. Compared to the PBS or the NC mice, levels of Bax mRNA and protein in tumor xenografts were significantly upregulated in p65 siRNA-treated mice. Knockdown of NF-κB p65 subunit expression significantly inhibited the growth of nude mouse Lewis tumor cell xenografts by induction of tumor cell apoptosis and significantly up-regulation of pro-apoptotic protein Bax expression. Future study will confirm the current data and targeting NF-κB p65 subunit expression as apotential therapeutic strategy in treating human lung cancer. NF-κB, lung cancer, apoptosis, small interfering RNA, xenografts.

Abstract 233: Prostacyclin analogs, iloprost and treprostinil, differentially influence proliferation of lung tumor cells

Abstract Lung cancer will kill approximately 160,000 Americans this year–more than the next four cancer subtypes (breast, prostate, colorectal, and pancreatic) combined. Even if lung cancer is diagnosed at an early stage, the 5 year survival rate is 52.2%. The poor survival rates for early stage disease suggests that chemopreventive interventions in high-risk populations are necessary. We found that prostacyclin (prostaglandin I2 or PGI2) synthase (PGIS) overexpression retards lung tumor growth in preclinical chemical carcinogen and smoking models, and treatment with iloprost (a PGI2 analog currently approved for treatment of pulmonary hypertension) inhibits lung adenocarcinoma formation in these same preclinical models. Oral iloprost also improves bronchial dysplasia (precursor lesions to squamous cell lung cancer) in former smokers. Mechanistic in vitro and in vivo studies using genetically modified mice showed that PGI2 and iloprost exert their chemopreventive effects through activation of the peroxisome proliferator-activated receptor γ (PPARγ) pathway rather than the classic PGI2 receptor, IP1, and that this activation requires frizzled 9 (Fzd9) activity. Treprostinil, a next generation PGI2 analog also approved to treat pulmonary hypertension, activates IP1 as well, but its ability to activate PPARγ/Fzd9 has yet to be fully characterized. In vitro studies show that in addition to binding to IP, iloprost and treprostinil differentially activate other prostaglandin (PG) receptors suggesting that they may have different chemopreventive efficacies. Molecular modeling of iloprost and treprostinil binding to PPARγ indicates these molecules may induce different PPARγ conformations which could affect binding of co-activators/repressors or interaction with the retinoic acid receptor (which is required for induction of gene transcription). While iloprost does not affect mouse lung tumor cell proliferation in vitro, treprostinil and prostaglandin E2 actually increase proliferation 2-5 fold in a dose dependent manner. Alveolar macrophage conditioned media also increases mouse lung tumor cell proliferation, and this increase is enhanced if macrophages are treated with treprostinil or PGE2 prior to media harvest. If macrophages are pretreated with iloprost, either alone or in combination with PGE2 or treprostinil, this increased proliferation is attenuated suggesting that iloprost may be exerting its chemopreventive effects directly on tumor cells and by modulating macrophage production of pro-growth factors. Macrophage IL-6 production decreased almost 80% with iloprost pre-treatment while little effect was seen with treprostinil pre-treatment. Comparisons between iloprost and treprostinil may indicate new agents/targets for lung cancer chemoprevention. Citation Format: Lori D. Dwyer-Nield, Gregory Hickey, Meredith A. Tennis, Kevin S. Choo, Donald S. Backos, Robert L. Keith. Prostacyclin analogs, iloprost and treprostinil, differentially influence proliferation of lung tumor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 233. doi:10.1158/1538-7445.AM2014-233

No Involvement of Lysophosphatidic Acid Receptor-3 in Cell Migration of Mouse Lung Tumor Cells Stimulated by 12-O-Tetradecanoylphorbol-13-acetate

The tumor promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulates cell migration of several tumor cells. Recently, we reported that loss of lysophosphatidic acid (LPA) receptor-3 (LPA3) enhanced cell migration of murine lung tumor LL/2 cells. In the present study, we investigated whether LPA3 is involved in cell migration of mouse lung tumor cells stimulated by TPA. Exogenous LPA3 gene (Lpar3)-expressing (LL/2-a3) cells and LL/2-AB cells as a vector control generated from LL/2 cells were used. In a cell migration assay, TPA treatment significantly stimulated cell migration of LL/2-AB and LL/2-a3 cells, while the cell migration abilities of LL/2-a3 were markedly lower than those of LL/2-AB cells. Using quantitative real-time reverse transcription (RT)-polymerase chain reaction (PCR) analysis, no effect of TPA treatment on the expression levels of LPA1, LPA2 and LPA3 genes was detected in either type of cells. These results suggest that the LPA3 may not be involved in the enhanced migration ability by TPA in mouse lung tumor cells.

Selective oncolytic effect of an attenuated Newcastle disease virus (NDV-HUJ) in lung tumors

Newcastle disease virus (NDV), an avian paramyxovirus, has a potential oncolytic effect that may be of significance in the treatment of a variety of cancer diseases. An attenuated lentogenic isolate of NDV (HUJ) demonstrated a selective cytopathic effect upon a panel of human and mouse lung tumor cells, as compared to human nontumorigenic lung cells. The virus-selective oncolytic effect is apoptosis dependent, and related to higher levels of viral transcription, translation and progeny virus formation. Furthermore, NDV-HUJ oncolytic activity is directed in-cis and not through induction of cytokines, that may act in-trans on neighboring cells. Development of primary lung tumors and of the consequent metastasis in mice inoculated with mouse lung tumor cells 3LL-D122 was decreased following treatment with NDV-HUJ. The preferential killing of the tumor cells is not due to a deficiency in the interferon (IFN) system, as expression of the IFN-beta gene, in the infected cells, is properly induced. Moreover, pretreatment with IFN effectively protected the tumor cells from the virus oncolytic effect. We conclude therefore, that NDV-HUJ should have a significant benefit in the treatment of lung cancer as well as other malignancies.

Wildtype Kras2 can inhibit lung carcinogenesis in mice.

Although the ras genes have long been established as proto-oncogenes, the dominant role of activated ras in cell transformation has been questioned. Previous studies have shown frequent loss of the wildtype Kras2 allele in both mouse and human lung adenocarcinomas. To address the possible tumor suppressor role of wildtype Kras2 in lung tumorigenesis, we have carried out a lung tumor bioassay in heterozygous Kras2-deficient mice. Mice with a heterozygous Kras2 deficiency were highly susceptible to the chemical induction of lung tumors when compared to wildtype mice. Activating Kras2 mutations were detected in all chemically induced lung tumors obtained from both wildtype and heterozygous Kras2-deficient mice. Furthermore, wildtype Kras2 inhibited colony formation and tumor development by transformed NIH/3T3 cells and a mouse lung tumor cell line containing an activated Kras2 allele. Allelic loss of wildtype Kras2 was found in 67% to 100% of chemically induced mouse lung adenocarcinomas that harbor a mutant Kras2 allele. Finally, an inverse correlation between the level of wildtype Kras2 expression and extracellular signal-regulated kinase (ERK) activity was observed in these cells. These data strongly suggest that wildtype Kras2 has tumor suppressor activity and is frequently lost during lung tumor progression.

Intercellular communication and carcinogenesis

Two types of intercellular communication (humoral and cell contact-mediated) are involved in control of cellular function in multicellular organisms, both of them mediated by membrane-embedded proteins. Involvement of aberrant humoral communication in carcinogenesis has been well documented and genes coding for some growth factors and their receptors have been classified as oncogenes. More recently, cell contact-mediated communication has been found to have an important role in carcinogenesis, and some genes coding for proteins involved in this type of communication appear to form a family of tumor-suppressor genes. Both homologous (among normal or (pre-)cancerous cells) as well as heterologous (between normal and (pre)cancerous cells) communications appear to play important roles in cell growth control. Gap junctional intercellular communication (GJIC) is the only means by which multicellular organisms can exchange low molecular weight signals directly from within one cell to the interior of neighboring cells. GJIC is altered by many tumor-promoting agents and in many human and rodent tumors. We have recently shown that liver tumor-promoting agents inhibit GJIC in the rat liver in vivo. Molecular mechanisms which could lead to aberrant GJIC include: (1) mutation of connexin genes; (2) reduced and/or aberrant expression of connexin mRNA; (3) aberrant localization of connexin proteins, i.e., intracytoplasmic rather than in the cytoplasmic membrane; and (4) modulation of connexin functions by other proteins, such as those involved in extracellular matrix and cell adhesion. Whilst mutations of the cx 32 gene appear to be rare in tumors, cx 37 gene mutations have been reported in a mouse lung tumor cell line. Our results suggest that aberrant connexin localization is rather common in cancer cells and that possible molecular mechanisms include aberrant phosphorylation of connexin proteins and lack of cell adhesion molecules. Studies on transfection of connexin genes into tumor cells suggest that certain connexin genes (e.g., cx 26, cx 43 and cx 32) act as tumor-suppressor genes.