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International audience; Combined treatment targeting mitochondria may improve the efficacy of lung cancer chemoprevention. Here, mitochondria-targeted honokiol (Mito-HNK), an inhibitor of mitochondrial complex I and STAT3 phosphorylation, and mitochondria-targeted lonidamine (Mito-LND), an inhibitor of mitochondrial complexes I/II and AKT/mTOR/p70S6K signaling, were evaluated for their combinational chemopreventive efficacy on mouse lung carcinogenesis. All chemopreventive treatments began one-week post-carcinogen treatment and continued daily for 24 weeks. No evidence of toxicity (including liver toxicity) was detected by monitoring serum levels of alanine aminotransferase and aspartate aminotransferase enzymes. Mito-HNK or Mito-LND treatment alone reduced tumor load by 56% and 48%, respectively, whereas the combination of Mito-HNK and Mito-LND reduced tumor load by 83%. To understand the potential mechanism(s) of action for the observed combinatorial effects, single-cell RNA sequencing was performed using mouse tumors treated with Mito-HNK, Mito-LND, and their combination. In lung tumor cells, Mito-HNK treatment blocked the expression of genes involved in mitochondrial complex ǀ, oxidative phosphorylation, glycolysis, and STAT3 signaling. Mito-LND inhibited the expression of genes for mitochondrial complexes I/II, oxidative phosphorylation, and AKT/mTOR/p70S6K signaling in lung tumor cells. In addition to these changes, a combination of Mito-HNK with Mito-LND decreased arginine and proline metabolism, N-glycan biosynthesis, and tryptophan metabolism in lung tumor cells. Our results demonstrate that Mito-LND enhanced the antitumor efficacy of Mito-HNK, where both compounds inhibited common targets (oxidative phosphorylation) as well as unique targets for each agent (STAT3 and mTOR signaling). Therefore, the combination of Mito-HNK with Mito-LND may present an effective strategy for lung cancer chemoprevention.

Bexarotene has shown inhibition of lung and mammary gland tumorigenesis in preclinical models and in clinical trials. The main side effects of orally administered bexarotene are hypertriglyceridemia and hypercholesterolemia. We previously demonstrated that aerosolized bexarotene administered by nasal inhalation has potent chemopreventive activity in a lung adenoma preclinical model without causing hypertriglyceridemia. To facilitate its future clinical translation, we modified the formula of the aerosolized bexarotene with a clinically relevant solvent system. This optimized aerosolized bexarotene formulation was tested against lung squamous cell carcinoma mouse model and lung adenocarcinoma mouse model and showed significant chemopreventive effect. This new formula did not cause visible signs of toxicity and did not increase plasma triglycerides or cholesterol. This aerosolized bexarotene was evenly distributed to the mouse lung parenchyma, and it modulated the microenvironment in vivo by increasing the tumor-infiltrating T cell population. RNA sequencing of the lung cancer cell lines demonstrated that multiple pathways are altered by bexarotene. For the first time, these studies demonstrate a new, clinically relevant aerosolized bexarotene formulation that exhibits preventive efficacy against the major subtypes of lung cancer. This approach could be a major advancement in lung cancer prevention for high risk populations, including former and present smokers.

Expressed on cells of the myeloid and lymphoid lineages, V-domain Ig Suppressor of T cell Activation (VISTA) is an emerging target for cancer immunotherapy. Blocking VISTA activates both innate and adaptive immunity to eradicate tumors in mice. Using a tripeptide small molecule antagonist of VISTA CA170, we found that it exhibited potent anticancer efficacy on carcinogen-induced mouse lung tumorigenesis. Remarkably, lung tumor development was almost completely suppressed when CA170 was combined with an MHCII-directed KRAS peptide vaccine. Flow cytometry and single-cell RNA sequencing (scRNA-seq) revealed that CA170 increased CD8+ T cell infiltration and enhanced their effector functions by decreasing the tumor infiltration of myeloid-derived suppressor cells (MDSCs) and Regulatory T (Treg) cells, while the Kras vaccine primarily induced expansion of CD4+ effector T cells. VISTA antagonism by CA170 revealed strong efficacy against lung tumorigenesis with broad immunoregulatory functions that influence effector, memory and regulatory T cells, and drives an adaptive T cell tumor-specific immune response that enhances the efficacy of the KRAS vaccine., Pan, Chen, Zhang et al. investigated the anti-cancer efficacy of CA170, a small molecule agonist of VISTA, on mouse lung tumorigenesis. When combined with a MHCII-directed KRAS peptide vaccine, the authors reported that CA170 suppressed lung tumor development by increasing CD8+ T cell and decreasing MDSC and Treg infiltration, while the Kras vaccine induced CD4+ effector T cell expansion

Summary: We synthesized a mitochondria-targeted honokiol (Mito-HNK) that facilitates its mitochondrial accumulation; this dramatically increases its potency and efficacy against highly metastatic lung cancer lines in vitro, and in orthotopic lung tumor xenografts and brain metastases in vivo. Mito-HNK is >100-fold more potent than HNK in inhibiting cell proliferation, inhibiting mitochondrial complex ǀ, stimulating reactive oxygen species generation, oxidizing mitochondrial peroxiredoxin-3, and suppressing the phosphorylation of mitoSTAT3. Within lung cancer brain metastases in mice, Mito-HNK induced the mediators of cell death and decreased the pathways that support invasion and proliferation. In contrast, in the non-malignant stroma, Mito-HNK suppressed pathways that support metastatic lesions, including those involved in inflammation and angiogenesis. Mito-HNK showed no toxicity and targets the metabolic vulnerabilities of primary and metastatic lung cancers. Its pronounced anti-invasive and anti-metastatic effects in the brain are particularly intriguing given the paucity of treatment options for such patients either alone or in combination with standard chemotherapeutics. : Natural Product Chemistry; Immunology; Medicinal and Aromatic Plants Subject Areas: Natural Product Chemistry, Immunology, Medicinal and Aromatic Plants

Lung squamous cell carcinoma (LUSC) is a major subtype of Non-Small Cell Lung Cancer. To increase our understanding of the LUSC pathobiology, we performed exome sequencing and RNA-seq in 16 murine carcinogen-induced LUSC tumors and 8 normal murine lung tissue samples. Additionally, we conducted single-cell RNA-seq on two independent tumors from the same murine model. We identified a list of 59 cancer genes recurrently mutated in the mice LUSC tumors, 47 (80%) of which were also mutated in human LUSCs. At the single cell level, we detected unique clonal mutation patterns for each of the two LUSC tumors, being initiated from clones carrying the mutant Igfbp7 and Trp53 genes, respectively. We also identified an expression signature serving as an effective classifier for LUSC tumors and a strong predictor of survival outcomes of lung cancer patients. Lastly, we found that some of the mutant LUSC genes were associated with the significantly altered tumoral expression of inhibitory immune checkpoint genes such as PD-L1, VISTA, TIM3 and LAG3 in human LUSCs. The novel findings of clonal evolution, mutational landscapes and expression signatures of LUSC suggested new targets for the overall LUSC therapy and the immunotherapy of LUSC.

Lung cancer is the leading cause of cancer mortality worldwide. More than 85% of lung cancers are non-small cell lung cancer (NSCLC). EGFR mutations occur in 47.9% of Asia-Pacific patients with NSCLC and 19.2% of Western patients. The most common EGFR mutations (>90%) are deletions in exon 19 and/or point mutations in exon 21 (L858R). Th1 helper cellular immunity is critical for immunotherapy-mediated tumor regression. We have previously characterized an MHC-II-restricted EGFR multi-peptide vaccine (two peptides: “SCVRACGADSYEMEEDGVRK” and “VWSYGVTVWELMTFGSKPY”) (EGFR-V) that targets the EGFR protein and decreases EGFR-driven lung tumorigenesis by ~80% in EGFRL858R transgenic mice that were vaccinated before doxycycline-induction of the EGFR protein [1]. However, diminished efficacy was observed when this MHC-II-restricted EGFR multi-peptide vaccine was given two weeks after doxycycline induction of the EGFR protein, suggesting that expression of the EGFR oncoprotein significantly increased the immunosuppressive microenvironment. JHU083, an orally bioavailable glutamine antagonist, has recently been shown to not only inhibit tumor growth but also boost anti-cancer immunity [2]. To determine if JHU083 could potentiate the efficacy of EGFR vaccine in a post-initiation setting, we evaluated in vivo antitumor efficacy of EGFR vaccine combined with JHU083 using an EGFRL858R transgenic mouse model. JHU083 inhibited tumor burden by 31% and EGFR vaccine suppressed tumor burden by 33%, whereas combining JHU083 with anti-EGFR peptide vaccine had an additive effect (54% tumor inhibition). Mechanistically, JHU083 by itself, markedly reduced the immunosuppressive monocytic myeloid-derived suppressor cells (MDSCs) in lung tissues. The combination of JHU083 and the vaccine significantly reduced Tregs in lung tissues. The anti-EGFR vaccine primarily induced expansion of antigen specific antitumor CD4+ effector T cells. Long term administration of JHU-083 did not decrease bodyweight in mice. Together with previous results, these data suggest that JHU083 could be used to reshape the tumor microenvironment toward one that enhances antitumor T cell responses and could further enhance the efficacy of the EGFR anticancer vaccine. References1.Ebben, J.D., et al., Epidermal growth factor receptor derived peptide vaccination to prevent lung adenocarcinoma formation: An in vivo study in a murine model of EGFR mutant lung cancer. Mol Carcinog, 2016. 55(11): p. 1517-1525.2.Leone, R.D., et al., Glutamine blockade induces divergent metabolic programs to overcome tumor immune evasion. Science, 2019. 366(6468): p. 1013-1021. Citation Format: Mofei Huang, Jing Pan, Qi Zhang, Shizuko Sei, Robert Shoemaker, Ronald Lubet, Yian Wang, Barbara Slusher, Ming You. Potentiation of EGFR peptide cancer vaccine by an orally bioavailable glutamine antagonist prodrug JHU-083 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 440.

// Qi Zhang 1, 2 , Ruichao Li 2 , Xu Chen 1, 2 , Sang Beom Lee 2 , Jing Pan 1, 2 , Donghai Xiong 1, 2 , Jiaqi Hu 2 , Mark Steven Miller 3 , Eva Szabo 3 , Ronald A. Lubet 3 , Yian Wang 1, 2 and Ming You 1, 2 1 Cancer Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA 2 Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, WI 53226, USA 3 Chemopreventive Agent Development Research Group, Division of Cancer Prevention, National Cancer Institute, Rockville, MD 20850, USA Correspondence to: Ming You, email: myou@mcw.edu Keywords: weekly, daily, intermittent, Gefitinib, lung cancer Received: February 16, 2017 Accepted: June 27, 2017 Published: August 02, 2017 ABSTRACT Gefitinib showed response in phase II clinical trials and with better clinical response in lung cancer with activating mutations in the tyrosine kinase domain of the EGFR. Questions of toxicity and potential dosing regimens impede the use in a prevention setting. This study will provide scientific evidence for the utility of testing and comparing weekly and daily dosing regimens in clinical trials. We employed the adenocarcinoma (AD) and squamous cell carcinoma (SCC) models to compare the efficacy of Gefitinib in daily or weekly dosing regimens. We also assessed the effectiveness of Gefitinib in altering growth of the H3255 xenograft. Bioluminescent imaging (BLI) and tumor size was evaluated. Relative expression of phospho-EGFR, phospho-ERK and phospho-AKT in the xenograft were evaluated by Western Blot analysis. In the lung AD model, Gefitinib showed significant inhibition of tumor load when treated with weekly or weekly intermittent dosing regimens in AJ/p53 val135/wt mice whereas a daily dosing regimen did not decrease the tumor load significantly. In the H3255-Luciferase xenograft model, weekly treatment demonstrated better inhibition than daily treatment. The weekly dosing regimen exhibited greater inhibition of phospho-EGFR, phospho-ERK and phospho-AKT than the daily dosing regimen, which may be correlated with the antitumor effects of the different dosing regimens. Weekly dosing with Gefitinib had similar or better efficacy than the daily dosing regimen in pre-clinical models of NSCLC. The data provide scientific evidences for the utility of testing and comparing weekly and intermittent dosing regimens in clinical trials.

Lung cancer is the leading cause of cancer death in the United States. Metastasis to lymph nodes and distal organs, especially brain, leads to severe complications and death. Preventing lung cancer development and metastases is an important strategy to reduce lung cancer mortality. Honokiol (HNK), a natural compound present in the extracts of magnolia bark, has a favorable bioavailability profile and recently has been shown to readily cross the blood–brain barrier. In the current study, we evaluated the antimetastatic effects of HNK in both the lymph node and brain mouse models of lung tumor metastasis. We tested the efficacy of HNK in preventing 18 H2030-BrM3 cell (brain-seeking human lung tumor cells) migration to lymph node or brain. In an orthotopic mouse model, HNK significantly decreased lung tumor growth compared with the vehicle control group. HNK also significantly reduced the incidence of lymph node metastasis and the weight of mediastinal lymph nodes. In a brain metastasis model, HNK inhibits metastasis of lung cancer cells to the brain to approximately one third of that observed in control mice. We analyzed HNK's mechanism of action, which indicated that its effect is mediated primarily by inhibiting the STAT3 pathway. HNK specifically inhibits STAT3 phosphorylation irrespective of the mutation status of EGFR, and knockdown of STAT3 abrogated both the antiproliferative and the antimetastatic effects of HNK. These observations suggest that HNK could provide novel chemopreventive or therapeutic options for preventing both lung tumor progression and lung cancer metastasis. Cancer Prev Res; 10(2); 133–41. ©2016 AACR.

// Donghai Xiong 1, 2, * , Jing Pan 1, 2, * , Qi Zhang 1, 2, * , Eva Szabo 3 , Mark Steven Miller 3 , Ronald A. Lubet 3 , Ming You 1, 2 , Yian Wang 1, 2 1 Cancer Center, Medical College of Wisconsin, WI 53226, USA 2 Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, WI 53226, USA 3 Chemopreventive Agent Development Research Group, Division of Cancer Prevention, National Cancer Institute, Rockville, MD 20850, USA * These authors contributed equally to this work Correspondence to: Ming You, email: myou@mcw.edu Yian Wang, email: yiwang@mcw.edu Keywords: AUC (area under the curve), GSEA (gene set enrichment analysis), IPA (Ingenuity Pathway Analysis), PPAR (peroxisome proliferator-activated receptor gamma), SCC (squamous cell carcinoma) Received: September 22, 2016 Accepted: November 07, 2016 Published: December 07, 2016 ABSTRACT Due to exposure to environmental toxicants, a “field cancerization” effect occurs in the lung resulting in the development of a field of initiated but morphologically normal appearing cells in the damaged epithelium of bronchial airways with dysregulated gene expression patterns. Using a mouse model of lung squamous cell carcinoma (SCC), we performed transcriptome sequencing (RNA-Seq) to profile bronchial airway gene expression and found activation of the PI3K and Myc signaling networks in cytologically normal bronchial airway epithelial cells of mice with preneopastic lung SCC lesions, which was reversed by treatment with the PI3K Inhibitor XL-147 and pioglitazone, respectively. Activated MYC signaling was also present in premalignant and tumor tissues from human lung SCC patients. In addition, we identified a key microRNA, mmu-miR-449c-5p, whose suppression significantly up-regulated Myc expression in the normal bronchial airway epithelial cells of mice with early stage SCC lesions. We developed a novel bronchial genomic classifier in mice and validated it in humans. In the classifier, Ppbp (pro-platelet basic protein) was overexpressed 115 fold in the bronchial airways of mice with preneoplastic lung SCC lesions. This is the first report that demonstrates Ppbp as a novel biomarker in the bronchial airway for lung cancer diagnosis.

Background: Chemopreventive agent (CPA) treatment is one of the main preventive options for lung cancer. However, few studies have been done on pharmacodynamic biomarkers of known CPAs for lung cancer. Materials and methods: In this study, we treated mouse models of lung squamous cell carcinoma with three different CPAs (MEK inhibitor: AZD6244, PI-3K inhibitor: XL-147 and glucocorticoid: Budesonide) and examined circulating exosomal miRNAs in the plasma of each mouse before and after treatment. Results: Compared to baselines, we found differentially expressed exosomal miRNAs after AZD6244 treatment (n = 8, FDR &lt, 0.05, n = 55, raw p-values &lt, 0.05), after XL-147 treatment (n = 4, FDR &lt, n = 26, raw p-values &lt, 0.05) and after Budesonide treatment (n = 1, FDR &lt, n = 36, raw p-values &lt, 0.05). In co-expression analysis, we found that modules of exosomal miRNAs reacted to CPA treatments differently. By variable selection, we identified 11, 9 and nine exosomal miRNAs as predictors for AZD6244, XL-147 and Budesonide treatment, respectively. Integrating all the results, we highlighted 4 miRNAs (mmu-miR-215-5p, mmu-miR-204-5p, mmu-miR-708-3p and mmu-miR-1298-5p) as the key for AZD6244 treatment, mmu-miR-23a-3p as key for XL-147 treatment, and mmu-miR-125a-5p and mmu-miR-16-5p as key for Budesonide treatment. Conclusions: This is the first study to use circulating exosomal miRNAs as pharmacodynamic biomarkers for CPA treatment in lung cancer.

Kirsten rat sarcoma viral oncogene homolog (KRAS) gene encodes a small GTPase that cycles between GDP and GTP-bound states, which modulates important cell surface receptors essential for cell proliferation and survival. It is one of the most common drivers for many types of cancers, is present in up to 25% all human tumors, and 80% of mutation occurs in codon12. Efforts to target KRAS-driven cancers preventively or therapeutically have been unsuccessful. We recently formulated a multi-peptide KRAS vaccine (KVAX), which targets both wild-type and G12D mutant forms of KRAS and has 100% sequence homology between human and mouse. KVAX elicited strong immunologic response and exhibited striking cancer preventive efficacy when administered prior to KRAS mutation induction. However, clinical high-risk individuals may already express mutant KRAS, which has known to promote an immunosuppressive environment. Therefore, we combined KVAX with avasimibe (AVA), an ACAT1 inhibitor that modulates lipid metabolism in T cells and facilitates better TCR/peptide/MHC interactions, in syngeneic lung cancer mouse model and genetically engineered mouse model, in which mutant KRAS initiated lung tumorigenesis before vaccination. We found that the combination significantly decreased the presence of regulatory T cells in the tumor microenvironment, facilitated CD8+ T cell infiltration in tumor sites, and ultimately led to enhanced anti-cancer efficacy. Using 10X Genomics, we performed single cell RNA-seq on lung tumors from mice treated with KVAX and AVA and found significant increases in APC monocytes and CD4+ Th1 cells, especially increases in the effector/memory T cell population. Meanwhile, KVAX also decreased Tregs and M2 macrophages. These studies include the evaluation of tumor-infiltrating immune cells by single-cell RNA-seq (scRNA-seq) and provide a more detailed mechanisms of action of KVAX alone or in combination with AVA in modulating the tumor immune microenvironment. Citation Format: Jing Pan, Qi Zhang, Katie Palen, Bryon Johnson, Li Wang, Shizuko Sei, Robert H. Shoemaker, Ronald A. Lubet, Yian Wang, Ming You. A novel combination of a multipeptide KRAS vaccine and an ACAT1 inhibitor to prevent lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4584.

As a consequence of exposure to environmental toxicants, a “field cancerization” effect occurs in the lung, resulting in the development of a field of initiated, but morphologically normal appearing cells within a damaged epithelium containing mutations in oncogene or tumor suppressor genes. Unlike humans, whose airway field of injury associated with lung cancer has long been investigated with airway brushings obtained via bronchoscopy, no methods are available for similar studies in the mouse due to the small size of the murine airways. In this protocol, we describe a detailed method for performing airway brushing from a live mouse, which enables repeated sampling from the same mouse and thus, mimicking the bronchoscopy protocol used in humans. Using this approach in the N-nitroso-tris-chloroethylurea (NTCU)-induced mouse lung squamous cell carcinoma (SCC) model, we isolated airway epithelial cells with intact cell membrane structure and then performed transcriptome sequencing (RNA-Seq). We found activation of the PI3K signaling network to be the most significant in cytologically normal bronchial airway epithelial cells of mice with preneoplastic lung SCC lesions. Prolonged exposure to NTCU also induced activation of NF-kappaB (NFƙB), the downstream pathway of PI3K; this NTCU-induced lung SCC progression can be reversed by blocking the NFƙB pathway.

Non-small cell lung cancer (NSCLC) patients with an epidermal growth factor receptor (EGFR) mutation have benefited from treatment of reversible EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib. Acquisition of a secondary mutation in EGFR T790M is the most common mechanism of resistance to first generation EGFR TKIs, resulting in therapeutic failure. Afatinib is a second generation of EGFR TKI that showed great efficacy against tumors bearing the EGFR T790M mutation, but it failed to show the improvement on overall survival of lung cancer patients with EGFR mutations possibly because of novel acquired resistance mechanisms. Currently, there are no therapeutic options available for lung cancer patients who develop acquired resistance to afatinib. To identify novel resistance mechanism(s) to afatinib, we developed afatinib resistant cell lines from a parental human-derived NSCLC cell line, H1975, harboring both EGFR L858R and T790M mutations. We found that activation of the insulin-like growth factor 1 receptor (IGF1R) signaling pathway contributes to afatinib resistance in NSCLC cells harboring the T790M mutation. IGF1R knockdown not only significantly sensitizes resistant cells to afatinib, but also induces apoptosis in afatinib resistance cells. In addition, combination treatment with afatinib and linsitinib shows more than additive effects on tumor growth in in vivo H1975 xenograft. Therefore, these finding suggest that IGF1R inhibition or combination of EGFR-IGF1R inhibition strategies would be potential ways to prevent or potentiate the effects of current therapeutic options to lung cancer patients demonstrating resistance to either first or second generation EGFR TKIs © 2015 Wiley Periodicals, Inc.

Traditional Chinese Medicine (TCM) plays an indispensable role in China for both cancer prevention and adjuvant treatment in platinum-based chemotherapy. A number of naturally occurring products from Chinese herbal extracts have shown inhibitory effects against carcinogenesis. One example is JinFuKang which is a mixture of 12 Chinese herbs and has been used widely in China for the prevention and treatment of lung cancer. JinFuKang has been shown to inhibit tumor growth in human lung and liver cancer xenograft mouse models. Among all the herbal agents that contain the Astragalus membranaceus (Fisch), JinFuKang was found to be effective in improving the 24-month survival of lung cancer patients in combination with platinum-based chemotherapy. This review article will discuss the application of JinFuKang to cancer prevention and treatment.

3-Bromopyruvate (3-BrPA) is an alkylating agent and a well-known inhibitor of energy metabolism. Rapamycin is an inhibitor of the serine/threonine protein kinase mTOR. Both 3-BrPA and rapamycin show chemopreventive efficacy in mouse models of lung cancer. Aerosol delivery of therapeutic drugs for lung cancer has been reported to be an effective route of delivery with little systemic distribution in humans. In this study, 3-BrPA and rapamycin were evaluated in combination for their preventive effects against lung cancer in mice by aerosol treatment, revealing a synergistic ability as measured by tumor multiplicity and tumor load compared treatment with either single-agent alone. No evidence of liver toxicity was detected by monitoring serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes. To understand the mechanism in vitro experiments were performed using human non–small cell lung cancer (NSCLC) cell lines. 3-BrPA and rapamycin also synergistically inhibited cell proliferation. Rapamycin alone blocked the mTOR signaling pathway, whereas 3-BrPA did not potentiate this effect. Given the known role of 3-BrPA as an inhibitor of glycolysis, we investigated mitochondrial bioenergetics changes in vitro in 3-BrPA–treated NSCLC cells. 3-BrPA significantly decreased glycolytic activity, which may be due to adenosine triphosphate (ATP) depletion and decreased expression of GAPDH. Our results demonstrate that rapamycin enhanced the antitumor efficacy of 3-BrPA, and that dual inhibition of mTOR signaling and glycolysis may be an effective therapeutic strategy for lung cancer chemoprevention. Cancer Prev Res; 8(4); 318–26. ©2015 AACR.

PARK2, a gene associated with Parkinson disease, is a tumor suppressor in human malignancies. Here, we show that c.823C>T (p.Arg275Trp), a germline mutation in PARK2, is present in a family with eight cases of lung cancer. The resulting amino acid change, p.Arg275Trp, is located in the highly conserved RING finger 1 domain of PARK2, which encodes an E3 ubiquitin ligase. Upon further analysis, the c.823C>T mutation was detected in three additional families affected by lung cancer. The effect size for PARK2 c.823C>T (odds ratio = 5.24) in white individuals was larger than those reported for variants from lung cancer genome-wide association studies. These data implicate this PARK2 germline mutation as a genetic susceptibility factor for lung cancer. Our results provide a rationale for further investigations of this specific mutation and gene for evaluation of the possibility of developing targeted therapies against lung cancer in individuals with PARK2 variants by compensating for the loss-of-function effect caused by the associated variation.

Inflammatory conditions of the lung such as chronic obstructive pulmonary disease (COPD) are known to increase lung cancer risk, particularly lung squamous cell carcinoma (LSCC). In the present study, we developed a mouse model of inflammation-driven LSCC that was induced by N-nitroso-trischloroethylurea (NTCU) and enhanced by lipopolysaccharide (LPS), a potent proinflammatory agent contained in tobacco and tobacco smoke, and determined the chemopreventive effects of BioResponse diindolylmethane (DIM) in the same model. Compared with mice treated with NTCU alone, mice treated with the combination of NTCU and LPS had a 9-fold increase in the number of bronchioles with LSCC. Also, compared with mice treated with LPS alone, mice treated with NTCU plus LPS showed significantly increased expression of the inflammatory cytokines IL1α, IL6, and TNFα (all three increased about 7-fold). Parallel to the increased cytokine gene expression, the NTCU plus LPS-treated group exhibited significantly enhanced activation of NF-κB, STAT3, ERK, p-38, and Akt, expression of p53, COX-2, and Mcl-1, and NF-κB- and STAT3-DNA binding in the lung. Dietary administration of DIM (10 μmol/g diet or 2,460 ppm) to mice treated with NTCU plus LPS reduced the incidence of LSCC by 2-fold, suppressed activation/expression of proinflammatory and procarcinogenic proteins and NF-κB- and STAT3-DNA binding, but not the expression of cytokines and p53. This study highlights the potential significance of our mouse model to identify promising drugs or dietary agents for the chemoprevention of human LSCC and that DIM is a very good candidate for clinical lung cancer chemoprevention trials. Cancer Prev Res; 8(1); 77–85. ©2014 AACR.

Airway epithelial cells are prone to the damage caused by lung cancer risk factors, such as cigarette smoking. Little is known about surrogate biomarkers in the bronchial airway epithelium that can be used to assess the effect of potential chemoprevention drugs on lung adenocarcinoma formation/progression. Pioglitazone has been suggested as a chemoprevention drug for lung cancer. To study the mechanisms underlying the role of pioglitazone in lung cancer prevention, we performed transcriptome sequencing (RNA-Seq) and found that Kras signaling was repressed by pioglitazone treatment in the airway epithelial cells of mice with lung adenocarcinoma (FDR q = 9.8E-04). It was also found that glucose metabolic pathways were elevated in the airway epithelium of mice with lung adenocarcinomas and inhibited by pioglitazone treatment (FDR q = 0.01). Downregulation of glucose metabolism genes was also observed in lung tumors of mice treated with pioglitazone. The high-risk expression signature of elevated glucose metabolism was associated with poor survival outcome in multiple lung adenocarcinoma patient populations (P values ranging from 1.0E-9 to 5.5E-5). Our results suggest that the role of pioglitazone in preventing lung adenocarcinoma may depend on inhibiting Kras signaling and glucose metabolism, which may serve as biomarkers of agent action in the airway epithelium.

Insulin-like growth factor-1 receptor (IGF-1R) is a transmembrane heterotetramer that is activated by Insulin-like growth factor 1 and is crucial for tumor transformation and survival of malignant cells. Importantly, IGF-1R overexpression has been reported in many different cancers, implicating this receptor as a potential target for anticancer therapy. Picropodophyllin (PPP) is a potent inhibitor of IGF-1R and has antitumor efficacy in several cancer types. However, the chemopreventive effect of PPP in lung tumorigenesis has not been investigated. In this study, we investigated the chemopreventive activity of PPP in a mouse lung tumor model. Benzo(a)pyrene was used to induce lung tumors, and PPP was given by nasal inhalation to female A/J mice. Lung tumorigenesis was assessed by tumor multiplicity and tumor load. PPP significantly decreased tumor multiplicity and tumor load. Tumor multiplicity and load were decreased by 52% and 78% respectively by 4 mg/ml aerosolized PPP. Pharmacokinetics analysis showed good bioavailability of PPP in lung and plasma. Treatment with PPP increased staining for cleaved caspase-3 and decreased Ki-67 in lung tumors, suggesting that the lung tumor inhibitory effects of PPP were partially through inhibition of proliferation and induction of apoptosis. In human lung cancer cell lines, PPP inhibited cell proliferation, and also inhibited phosphorylation of IGF-1R downstream targets, AKT and MAPK, ultimately resulting in increased apoptosis. PPP also reduced cell invasion in lung cancer cell lines. In view of our data, PPP merits further investigation as a promising chemopreventive agent for human lung cancer. © 2014 Wiley Periodicals, Inc.

// Jing Pan 1, * , Qi Zhang 1, * , Shizuko Sei 2 , Robert H. Shoemaker 2 , Ronald A. Lubet 2 , Yian Wang 1 and Ming You 1 1 Cancer Center and Department of Pharmacology & Toxicology, Medical College of Wisconsin, Milwaukee, WI, USA 2 Chemopreventive Agent Development Research Group, Division of Cancer Prevention, National Cancer Institute, Bethesda, MD, USA * These authors have contributed equally to this work Correspondence to: Ming You, email: myou@mcw.edu Keywords: immunoprevention, KRAS, lung adenocarcinoma, peptide vaccine, MHC class II Received: December 06, 2016 Accepted: March 01, 2017 Published: August 01, 2017 ABSTRACT Lung cancer remains the leading cause of cancer death worldwide. Mutations in KRAS are detected in up to 30% of lung cancer cases. No effective therapies specifically targeting mutant KRAS have been developed. Vaccination against KRAS mutants is one of the venues of active exploration. The present study evaluated both immunogenicity and antitumor efficacy of a newly formulated multipeptide vaccine targeting multiple epitopes of the KRAS molecule. The formulated vaccine contained top four peptides, which elicited the strongest immunologic response and showed 100% sequence homology between human and mouse. The multipeptide KRAS vaccine was tested in an inducible CCSP-TetO-KRAS G12D mouse model, where the vaccine was administered prior to activating the mutant KRAS protein. The KRAS peptide vaccine exhibited striking efficacy, reducing tumor number and tumor burden by >80% when compared with adjuvant alone. Splenocytes collected from vaccinated animals showed a robust immunologic response to the immunizing peptides. Furthermore, in vitro stimulation of these splenocytes by the vaccinated peptides resulted in the secretion of cytokines indicative of Th1 responses but with minimal secretion of Th2-related cytokines. The multipeptide KRAS vaccine was immunogenic and efficacious in the primary prevention of KRAS-induced lung cancer, indicating that the approach potentially can be used to prevent other KRAS-driven cancers, either alone or in combination with other modalities.

Lung cancer is the leading cause of cancer-related death, with non-small cell lung cancer (NSCLC) being the predominant form of the disease. Most lung cancer is caused by the accumulation of genomic alterations due to tobacco exposure. To uncover its mutational landscape, we performed whole-exome sequencing in 31 NSCLCs and their matched normal tissue samples. We identified both common and unique mutation spectra and pathway activation in lung adenocarcinomas and squamous cell carcinomas, two major histologies in NSCLC. In addition to identifying previously known lung cancer genes (TP53, KRAS, EGFR, CDKN2A and RB1), the analysis revealed many genes not previously implicated in this malignancy. Notably, a novel gene CSMD3 was identified as the second most frequently mutated gene (next to TP53) in lung cancer. We further demonstrated that loss of CSMD3 results in increased proliferation of airway epithelial cells. The study provides unprecedented insights into mutational processes, cellular pathways and gene networks associated with lung cancer. Of potential immediate clinical relevance, several highly mutated genes identified in our study are promising druggable targets in cancer therapy including ALK, CTNNA3, DCC, MLL3, PCDHIIX, PIK3C2B, PIK3CG and ROCK2.

About 30% stage I non-small cell lung cancer (NSCLC) patients undergoing resection will recur. Robust prognostic markers are required to better manage therapy options. MicroRNAs (miRNAs) are a class of small non-coding RNAs of 19-25 nt and play important roles in gene regulation in human cancers. The purpose of this study is to identify miRNA expression profiles that would better predict prognosis of stage I NSCLC. MiRNAs extracted from 527 stage I NSCLC patients were profiled on the human miRNA expression profiling v2 panel (Illumina). The expression profiles were analyzed for their association with cancer subtypes, lung cancer brain metastasis and recurrence/relapse free survival (RFS). MiRNA expression patterns between lung adenocarcinoma and squamous cell carcinoma differed significantly with 171 miRNAs, including Let-7 family members and miR-205. Ten miRNAs associated with brain metastasis were identified including miR-145*, which inhibit cell invasion and metastasis. Two miRNA signatures that are highly predictive of RFS were identified. The first contained 34 miRNAs derived from 357 stage I NSCLC patients independent of cancer subtype, whereas the second containing 27 miRNAs was adenocarcinoma specific. Both signatures were validated using formalin-fixed paraffin embedded and/or fresh frozen tissues in independent data set with 170 stage I patients. Our findings have important prognostic or therapeutic implications for the management of stage I lung cancer patients. The identified miRNAs hold great potential as targets for histology-specific treatment or prevention and treatment of recurrent disease.

Prior studies have shown the retinoid X receptor (RXR) agonist bexarotene has preventive efficacy in rodent models of mammary and lung tumorigenesis albeit causing hypertriglyceridemia and hypercholesterolemia. We reasoned that bexarotene delivered by inhalation may provide sufficient dose directly to the respiratory tract to achieve efficacy while avoiding these side effects. In this study, the chemopreventive activity of aerosolized bexarotene was investigated in the benzo(a)pyrene [B(a)P]-induced mouse lung tumor model as assessed by tumor multiplicity and tumor load. Aerosolized bexarotene significantly decreased tumor multiplicity and tumor load by 43% and 74%, respectively. Our data showed that bexarotene can both inhibit proliferation and promote apoptosis in vivo. Our data also show that aerosolized bexarotene did not increase plasma total cholesterol and triglyceride level compared with diet group. These results indicate that aerosolization may be a safe and effective route of administering bexarotene for chemoprevention of lung cancer. Cancer Prev Res; 4(2); 270–6. ©2010 AACR.

Background: Genetic factors play important roles in lung cancer susceptibility. In this study, we replicated the association of 5p15.33 and 6p21.33 with familial lung cancer. Taking into account the previously identified genetic susceptibility variants on 6q23-25/RGS17 and 15q24-25.1, we further determined the cumulative association of these four genetic regions and the population attributable risk percent of familial lung cancer they account for. Methods: One hundred ninety-four case patients and 219 cancer-free control subjects from the Genetic Epidemiology of Lung Cancer Consortium were used for the association analysis. Each familial case was chosen from one high-risk lung cancer family that has three or more affected members. Single nucleotide polymorphisms (SNP) on chromosomal regions 5p15.33, 6p21.33, 6q23-25/RGS17, and 15q24-25.1 were assessed for their associations with familial lung cancer. The cumulative association of the four chromosomal regions with familial lung cancer was evaluated with the use of a linear logistic model. Population attributable risk percent was calculated for each SNP using risk ratio. Results: SNP rs31489 showed the strongest evidence of familial lung cancer association on 5p15.33 (P = 2 × 10−4; odds ratio, 0.57; 95% confidence interval, 0.42-0.77), whereas rs3117582 showed a weak association on 6p21.33 (P = 0.09; odds ratio, 1.47; 95% confidence interval, 0.94-2.31). Analysis of a combination of SNPs from the four regions provided a stronger cumulative association with familial lung cancer (P = 6.70 × 10−6) than any individual SNPs. The risk of lung cancer was increased to 3- to 11-fold among those subjects who had at least one copy of risk allele at each region compared with subjects without any of the risk factors. These four genetic regions contribute to a total of 34.6% of familial lung cancer in smokers. Conclusions: The SNPs in four chromosomal regions have a cumulative and significant association with familial lung cancer and account for about one-third of the population attributable risk for familial lung cancer. Cancer Epidemiol Biomarkers Prev; 19(2); 517–24

To compare the chemopreventive efficacy of Polyphenon E (Poly E), (−)-epigallocatechin-3-gallate (EGCG) and Polyphenon E without EGCG (Poly E-EGCG) on the development of benzo(a)pyrene (B(a)P)-induced lung tumors in A/J mice. Female A/J mice were given a single intraperitoneal injection of B(a)P (100 mg/kg body weight). One week after B(a)P injection, animals received AIN-76A purified powder diet containing 0.975% (wt/wt) EGCG, 0.525% (wt/wt) Poly E-EGCG or 1.5% (wt/wt) Poly E for 24 weeks or control diet with no additives. Poly E treatment significantly decreased tumor multiplicity by 52% and tumor load by 64%, while EGCG and Poly E-EGCG did not significantly inhibit lung tumor multiplicity. EGCG was more stable in a complex mixture (Poly E) than as a pure compound. EGCG was ineffective when administered by diet likely due to its instability. Thus, EGCG’s efficacy on mice lung tumorigenesis requires the presence of other tea catechins.

Lung cancer is the leading cause of cancer death worldwide. Here, we describe a genome-wide association study of chemically induced lung tumorigenesis on 593 mice from 21 inbred strains using 115,904 genotyped and 1,952,918 imputed single nucleotide polymorphisms (SNPs). Using a genetic background–controlled genome search, we identified a novel lung tumor susceptibility gene Las2 (Lung adenoma susceptibility 2) on distal chromosome 18. Las2 showed strong association with resistance to tumor induction (rs30245983; P = 1.87 × 10−9) as well as epistatic interactions (P = 1.71 × 10−3) with the pulmonary adenoma susceptibility 1 locus, a major locus affecting mouse lung tumor development (rs13459098, P = 5.64 × 10−27). Sequencing analysis revealed four nonsynonymous SNPs and two insertions/deletions in the susceptible allele of Las2, resulting in the loss of tumor suppressor activities in both cell colony formation and nude mouse tumorigenicity assays. Deletion of LAS2 was observed in ∼40% of human lung adenocarcinomas, implying that loss of function of LAS2 may be a key step for lung tumorigenesis. [Cancer Res 2009;69(15):6290–8]

Purpose: We have previously mapped a major susceptibility locus influencing familial lung cancer risk to chromosome 6q23-25. However, the causal gene at this locus remains undeter- mined. In this study, we further refined this locus to identify a single candidate gene, by fine mapping using microsatellite markers and association studies using high-density single nucleotide polymorphisms (SNP). Experimental Design: Six multigenerational families with five or more affected members were chosen for fine-mapping the 6q linkage region using microsatellite markers. For association mapping, we genotyped 24 6q-linked cases and 72 unrelated noncancer controls from the Genetic Epidemiology ofLung Cancer Consortium resources using the Affymetrix 500K chipset. Significant associations were validated in two independent familial lung cancer populations: 226 familial lung cases and 313 controls from the Genetic Epidemiology of Lung Cancer Consortium, and154 familial cases and 325 controls from Mayo Clinic. Each familial case was chosen from one high-risk lung cancer family that has three or more affected members. Results: A region-wide scan across 6q23-25 found significant association between lung cancer susceptibility and three single nucleotide polymorphisms in the first intron of the RGS17 gene. This association was further confirmed in two independent familial lung cancer populations. By quantitative real-time PCR analysis ofmatched tumor and normal human tissues, we f that RGS17 transcript accumulation is highly and consistently increased in sporadic lung cancers. Human lung tumor cell proliferation and tumorigenesis in nude mice are inhibited upon knockdown of RGS17 levels. Conclusion: RGS17 is a major candidate for the familial lung cancer susceptibility locus on chromosome 6q23-25. Lung cancer can occur sporadically in people with no known family history of lung cancer or it can be familial, occurring in multiple members of the same family. Initial evidence of a genetic basis for susceptibility to lung cancer came from

Three recent genome-wide association studies identified associations between markers in the chromosomal region 15q24-25.1 and the risk of lung cancer. We conducted a genome-wide association analysis to investigate associations between single-nucleotide polymorphisms (SNPs) and the risk of lung cancer, in which we used blood DNA from 194 case patients with familial lung cancer and 219 cancer-free control subjects. We identified associations between common sequence variants at 15q24-25.1 (that spanned LOC123688 [a hypothetical gene], PSMA4, CHRNA3, CHRNA5, and CHRNB4) and lung cancer. The risk of lung cancer was more than fivefold higher among those subjects who had both a family history of lung cancer and two copies of high-risk alleles rs8034191 (odds ratio [OR] = 7.20, 95% confidence interval [CI] = 2.21 to 23.37) or rs1051730 (OR = 5.67, CI = 2.21 to 14.60, both of which were located in the 15q24-25.1 locus, than among control subjects. Thus, further research to elucidate causal variants in the 15q24-25.1 locus that are associated with lung cancer is warranted.

Effective chemoprevention of lung cancer in high-risk patients through the administration of pharmacologic or nutritional agents is urgently needed. Aerosol inhalation can deliver chemopreventive agents directly to the respiratory tract to inhibit the tumorigenic process. In this study, polyphenon E (PolyE) and (-)-epigallocatechin-3-gallate (EGCG) were administered by aerosol delivery to A/J mice beginning 2 weeks after carcinogen treatment and continuing daily by inhalation throughout the remainder of the study (20 weeks). PolyE decreased tumor load by approximately 59%. However, EGCG, both at the same dose and at a higher dose, failed to inhibit lung carcinogenesis. These results indicate that aerosol delivery of PolyE, but not EGCG, may be a useful chemopreventive protocol in subjects at high risk for lung cancer.

In this study, we observed loss of heterozygosity (LOH) in human chromosomal fragment 6q25.1 in sporadic lung cancer patients. LOH was observed in 65% of the 26 lung tumors examined and was narrowed down to a 2.2-Mb region. Single-nucleotide polymorphism (SNP) analysis of genes located within this region identified a candidate gene, termed p34. This gene, also designated as ZC3H12D, C6orf95, FLJ46041, or dJ281H8.1, carries an A/G nonsynonymous SNP at codon 106, which alters the amino acid from lysine to arginine. Nearly 73% of heterozygous lung cancer tissues with LOH and the A/G SNP also exhibited loss of the A allele. In vitro clonogenic and in vivo nude mouse studies showed that overexpression of the A allele exerts tumor suppressor function compared with the G allele. p34 is located within a recently mapped human lung cancer susceptibility locus, and association of the p34 A/G SNP was tested among these families. No significant association between the less frequent G allele and lung cancer susceptibility was found. Our results suggest that p34 may be a novel tumor suppressor gene involved in sporadic lung cancer but it seems not to be the candidate familial lung cancer susceptibility gene linked to chromosomal region 6q23-25. [Cancer Res 2007;67(1):93–9]

Although ras and p53 are the most commonly found oncogene and tumor suppressor gene, respectively, in human cancers, their collective roles in tumor progression have yet to be defined in animal models. Here, we demonstrated the synergistic effect between ras and p53 in promoting tumor progression during lung tumorigenesis using bitransgenic mice. Mice with a heterozygous knockout of K-ras (K-ras(wt/ko)) were mated to p53 transgenic mice (p53(val135/wt)) in lung tumorigenesis (K-ras(wt/ko) x p53(val135/wt)). F(1) mice exhibited a significant increase in lung tumor load (tumor multiplicity x tumor volume) when compared to those seen in either K-ras(wt/ko) mice or p53(val135/wt) mice alone. Furthermore, over 50% of the lung tumors were lung adenocarcinomas in bitransgenic mice compared to only 3% in wild-type mice. Alterations of ras and p53 appear to promote the development of lung adenocarcinomas. These results provide the in vivo experimental evidence of synergistic interactions of ras and p53 in lung tumor progression.

Bexarotene (Targretin®, Ligand Pharmaceuticals Inc.) is a synthetic high-affinity RXR receptor agonist with limited affinity for RAR receptors. Bexarotene has shown efficacy in a phase I/II trial of non-small-cell lung cancers. However, the chemopreventive efficacy of bexarotene has not been determined in mouse lung cancer models. In this study, we have investigated the ability of bexarotene to inhibit lung tumor progression in the mutant A/J mouse models with genetic alterations in p53 or K-ras, two of the most commonly altered genes in human lung tumorigenesis. Mice were administered vinyl carbamate (VC), a carcinogen, by a single intraperitoneal injection (i.p.) at 6 weeks of age. Bexarotene was given by gavage starting at 16 weeks after VC and was continued for 12 weeks. Although all mice developed lung tumors, only 7% of lung tumors were adenocarcinomas in wild-type mice, whereas 22 and 26% of lung tumors were adenocarcinomas in p53 transgenic or K-ras heterozygous deficient mice. Bexarotene inhibited both tumor multiplicity and tumor volume in mice of all three genotypes. Furthermore, bexarotene reduced the progression of adenoma to adenocarcinoma by ∼50% in both p53wt/wtK-rasko/wt and p53wt/wtK-raswt/wt mice. Thus, bexarotene appears to be an effective preventive agent against lung tumor growth and progression.

Antitumor B (ATB), also known as Zeng Sheng Ping, is a Chinese herbal mixture composed of six plants. Previously, clinical studies have shown a significant chemopreventive efficacy of ATB against human esophageal and lung cancers. In the present study, A/J mice harboring a dominant-negative p53 and/or heterozygous deletion of Ink4a/Arf and treated with benzo[a]pyrene were used to investigate the chemopreventive effects of ATB on chemically induced lung tumorigenesis. Mice with various genotypes treated with ATB displayed a significant reduction in lung tumor multiplicity and tumor load. Treatment with ATB resulted in an approximately 40% decrease in tumor multiplicity and a 70% decrease in tumor load in both wild-type mice and in mice with a loss of the Ink4a/Arf tumor suppressor genes. Interestingly, ATB decreased tumor multiplicity and volume by 50 and 90%, respectively, in mice with a dominant-negative p53 and in mice with both a p53 mutation and deletion of Ink4a/Arf. Kras2 mutation analysis of the lung tumors revealed that tumors harbored mutations in the 12th codon of Kras2. There were no differences in either the incidence or types of mutations between tumors treated with or without ATB. Oligonucleotide array analysis revealed 284 genes that were differentially expressed in mouse lung tumors as compared to the normal lung, and it was found that 114 out of these 284 genes changed their expression toward the normal levels in tumors treated with ATB. Most of the genes modulated by ATB belong to several cellular signaling pathways, including Notch (Notch homolog 2, manic fringe homolog), growth factor (FGF intracellular-binding protein, PDGFalpha), G protein-Ras-MAPK (MAPK3, MAP3K4, rab3A, Rap1, RSG5, PKCtheta), ubiquitin-proteasome (CDC34, Cullin1, 26S proteasome), and apoptosis (BAD promoter, caspase 3). These results suggest that ATB is an effective chemopreventive against mouse lung tumorigenesis. Furthermore, ATB exhibited an enhanced inhibitory effect in animals harboring genetic alterations (Kras2, p53, and Ink4a/Arf), which are often seen in human lung adenocarcinomas.

Lung cancer, primarily associated with tobacco use, is the leading cause of cancer morbidity and mortality in the United States. Squamous cell carcinoma (SCC) is one of the four major histological types of lung cancer. Although there are several established models for lung adenoma and adenocarcinomas, there is no well-established mouse model for lung SCC. We treated eight different inbred strains of mice with N-nitroso-tris-chloroethylurea by skin painting and found that this regimen induced lung SCCs in five strains of mouse (SWR/J, NIH Swiss, A/J, BALB/cJ, and FVB/J) but not in the others (AKR/J, 129/svJ, and C57BL/6J). Mouse lung SCCs have similar histopathological features and keratin staining to human SCC. Moreover, a wide spectrum of abnormal lung squamous phenotypes including hyperplasia, metaplasia, carcinoma in situ, and invasive carcinoma, were observed. There are strain-specific differences in susceptibility to Lscc induction by N-nitroso-tris-chloroethylurea with NIH Swiss, A/J, and SWR/J mice developing scores of SCCs whereas the resistant strains AKR/J, 129/svJ, and C57BL/6J failed to develop any SCCs. FVB/J and BALB/cJ mice had an intermediate response. We conducted whole-genome linkage disequilibrium analysis in seven strains of mice, divided into three phenotype categories of susceptibility, using Fisher’s exact test applied to 6,128 markers in publically available databases. Three markers were found significantly associated with susceptibility to SCC with the P < 0.05. They were D1Mit169, D3Mit178, and D18Mit91. Interestingly, none of these sites overlap with the major susceptibility loci associated with lung adenoma/adenocarcinoma development in mice. The mouse SCC described here is highly significant for preclinical studies of lung cancer chemopreventive agents because most human trials have been conducted against precancerous lesions for SCC. Furthermore, this model can be used in determining genetic modifiers that contribute to susceptibility or resistance to lung SCC development.

Mutations in the Kras2 gene are seen in both human and mouse lung adenocarcinomas. The protein product (p21ras) encoded by the Kras2 gene must be post-translationally modified at a terminal CAAX motif in order to be biologically active. In this study, we systematically investigated the chemopreventive efficacy of two different farnesyltransferase inhibitors (FTIs): one is a peptidomimetic (FTI-276) and the other is an imidazole (L778-123). Both FTIs are designed to inhibit the post-translational modification of p21ras proteins with a terminal CAAX motif. In a complete chemoprevention study, where the inhibitor was administered before carcinogen was given, and throughout the study, FTI-276 treatment significantly reduced both the tumor multiplicity by 41.7% (P

Previous observation has shown that the wild-type Kras2 allele is a suppressor of lung cancer in mice. Here we report that loss of heterozygosity (LOH) of chromosome 12p was detected in approximately 50% of human lung adenocarcinomas and large cell carcinomas, and Kras2 mutations were detected at codon 12 in approximately 40% of adenocarcinomas and large cell carcinomas. Interestingly, all of the lung adenocarcinomas and large cell carcinomas containing a Kras2 mutation exhibited allelic loss of the wild-type Kras2 allele when a correlation between LOH of the region on chromosome 12p and Kras2 mutation was made. These results from human lung cancer tissues provide a strong evidence in support of our previous observation in mouse models that the wild-type Kras2 is a tumor suppressor of lung cancer.

Because of similarities in histopathology and tumor progression stages between mouse and human lung adenocarcinomas, the mouse lung tumor model with lung adenomas as the endpoint has been used extensively to evaluate the efficacy of putative lung cancer chemopreventive agents. In this study, a competitive cDNA library screening (CCLS) was employed to determine changes in the expression of mRNA in chemically induced lung adenomas compared with paired normal lung tissues. A total of 2555 clones having altered expression in tumors were observed following competitive hybridization between normal lung and lung adenomas after primary screening of over 160,000 clones from a mouse lung cDNA library. Among the 755 clones confirmed by dot blot hybridization, 240 clones were underexpressed, whereas 515 clones were overexpressed in tumors. Sixty-five clones with the most frequently altered expression in six individual tumors were confirmed by semiquantitative RT-PCR. When examining the 58 known genes, 39 clones had increased expression and 19 had decreased expression, whereas the 7 novel genes showed overexpression. A high percentage (>60%) of overexpressed or underexpressed genes was observed in at least two or three of the lesions. Reproducibly overexpressed genes included ERK-1, JAK-1, surfactant proteins A, B, and C, NFAT1, α-1 protease inhibitor, helix-loop-helix ubiquitous kinase (CHUK), α-adaptin, α-1 PI2, thioether S-methyltransferase, and CYP2C40. Reproducibly underexpressed genes included paroxanase, ALDH II, CC10, von Ebner salivary gland protein, and α- and β-globin. In addition, CCLS identified several novel genes or genes not previously associated with lung carcinogenesis, including a hypothetical protein (FLJ11240) and a guanine nucleotide exchange factor homologue. This study shows the efficacy of this methodology for identifying genes with altered expression. These genes may prove to be helpful in our understanding of the genetic basis of lung carcinogenesis and in developing biomarkers for lung cancer chemoprevention studies in mice.

Lung cancer remains the leading cause of cancer death worldwide. Mutations in KRAS are detected in up to 30% of lung cancer cases, about 80% of which occur in codon 12, yet no effective therapies specifically targeting mutant KRAS have been developed. Boosting the host immune response to cancer cells by vaccinating against defined tumor-associated antigens (TAAs) as a means of treating established tumors or preventing the development of tumors in high risk individuals such as current or former smokers is an area of intense research. The present study evaluated both immunogenicity and anti-tumor efficacy of a newly formulated multipeptide (peptides 15-17 amino acids long) vaccine targeting multiple epitopes of the KRAS molecule in a mouse model of KRAS-driven lung tumor. Vaccination was performed in the prevention setting in a transgenic mouse model, where mutated mouse Kras (G12D) is conditionally over-expressed in the lungs of mice using a CCSP promoter. A multi-score prediction algorithm was used to identify likely immunogenic epitopes in the KRAS protein sequence. The identified peptides were synthesized and screened in naïve mice for immunogenicity. The formulated vaccine contained the top four peptides, which elicited the strongest immunologic response and showed 100% sequence homology between human and mouse. The multipeptide KRAS vaccine was then tested in an inducible CCSP-TetO-KRASG12D mouse model, where the vaccines mixed with the Freund’s adjuvant or adjuvant alone were administered prior to activating the mutant KRAS protein. Our results show the KRAS peptide vaccine exhibited striking efficacy, reducing tumor number and tumor burden by >80% when compared with adjuvant alone. Splenocytes collected from vaccinated animals at the end of the study showed a robust immunologic response to the immunizing peptides. Furthermore, in vitro stimulation of these splenocytes by the vaccine peptides resulted in the secretion of cytokines indicative of Th1 (interferon gamma) and Th17 (IL17) responses but with minimal secretion of Th2 related cytokines e.g. IL3 and IL4. In the draining lymph nodes from vaccinated mice rejecting tumors, there was a substantial increase in CD4 cells but a limited increase in CD8 cells. In summary, the multipeptide KRAS vaccine was immunogenic and efficacious in the primary prevention of KRAS-induced lung cancer in mice, indicating that the approach can potentially be used for the prevention of KRAS-driven cancers either alone or in combination with other modalities. Citation Format: Jing Pan, Qi Zhang, Shizuko Sei, Robert H. Shoemaker, Ronald A. Lubet, Yian Wang, Ming You. Immunoprevention of KRAS-driven lung adenocarcinoma by a multi-peptide KRAS vaccine [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 1698. doi:10.1158/1538-7445.AM2017-1698

Lung cancer is the leading cause of cancer-related deaths in the United States. The 5 year survival rate for lung cancer patients has remained a dismal 15% for 3 decades. Thus, the development of chemopreventive agents that could prevent lung cancer could potentially reduce the incidence and mortality of pulmonary neoplasms. Bexarotene has exhibited inhibitory effects in preclinical in vivo models of mammary and lung tumorigenesis, has been approved for clinical use in the treatment of cutaneous T-cell lymphoma, and has shown efficacy in phase I/II trials of non-small cell lung cancer (NSCLC). Preclinical studies have demonstrated that it is highly effective in the prevention of all three major subtypes of lung cancer in mouse models: adenocarcinoma (AD), squamous cell carcinoma (SCC), and small cell lung cancer (SCLC). The major side effects of bexarotene when administered orally to rodents or human patients have been hypertriglyceridemia and hypercholesterolemia. Previous studies in a mouse model of lung AD have demonstrated that aerosol delivery of bexarotene through nasal inhalation exhibited potent chemopreventive activity similar to that observed following oral administration (59 to74% reductions in lung tumor multiplicity). The significant decreases in tumor multiplicity and tumor load were achieved without hypertriglycerides that accompany oral bexarotene administration. In this study, aerosolized delivery of 10-30 mg/ml bexarotene showed a significant chemopreventive effect in all three major subtypes. In the N-nitroso-tris-chloroethylurea (NTCU) induced mouse SCC model, 1 week after the first dose of NTCU a dose dependent decrease in tumor formation was observed, with the highest dose causing 75% (p Citation Format: Qi Zhang, Jing Pan, Marker S. Miller, Ronald A. Lubet, Yian Wang, Ming You. Preventive effect of aerosolized bexarotene in three major subtypes of lung cancer: adenocarcinoma, squamous cell carcinoma and small cell lung cancer in mice [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 188. doi:10.1158/1538-7445.AM2017-188

This study was designed to test the chemopreventive potential of perillyl alcohol, an inhibitor of farnesyltransferase, in a mouse lung tumor bioassay. Perillyl alcohol is a naturally occurring monoterpene found in lavender, cherries, and mint. We have shown previously that the majority of lung tumors in this bioassay have an activating mutation in the K-ras gene, which occurs early in the development of mouse lung carcinogenesis. The Ras protein undergoes a series of post-translational modifications, the first of which is farnesylation at the cysteine of the C-terminal CAAX motif. These modifications lead to the anchoring of Ras p21 to the plasma membrane in its biologically active state. Activated Ras p21 couples growth regulatory signals from receptor tyrosine kinases to cytoplasmic second messengers. In a preliminary study, we determined the maximum tolerated dose of perillyl alcohol to be 75 mg/kg body weight. For the bioassay, 5-week-old male (C3H/HeJ X A/J) F1 hybrid mice were randomized into trial groups, and treated with perillyl alcohol three times per week i.p., starting 1 week prior to initiation with the carcinogen NNK, and continuing for 22 weeks after initiation. Our results show a 22% reduction in tumor incidence, and a 58% reduction in tumor multiplicity. Our study demonstrates that perillyl alcohol is an effective chemopreventive compound in the mouse lung tumor bioassay.