Ying Yan

A chemically induced model for squamous cell carcinoma of the lung in mice: histopathology and strain susceptibility.

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.

Cancer chemopreventive activity of a mixture of Chinese herbs (antitumor B) in mouse lung tumor models

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, PDGFα), G protein-Ras-MAPK (MAPK3, MAP3K4, rab3A, Rap1, RSG5, PKCθ), 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.

A Gene Expression Signature that Can Predict Green Tea Exposure and Chemopreventive Efficacy of Lung Cancer in Mice

Green tea has been shown to be a potent chemopreventive agent against lung tumorigenesis in animal models. Previously, we found that treatment of A/J mice with either green tea (0.6% in water) or a defined green tea catechin extract (polyphenon E; 2.0 g/kg in diet) inhibited lung tumor tumorigenesis. Here, we described expression profiling of lung tissues derived from these studies to determine the gene expression signature that can predict the exposure and efficacy of green tea in mice. We first profiled global gene expressions in normal lungs versus lung tumors to determine genes which might be associated with the tumorigenic process (TUM genes). Gene expression in control tumors and green tea-treated tumors (either green tea or polyphenon E) were compared to determine those TUM genes whose expression levels in green tea-treated tumors returned to levels seen in normal lungs. We established a 17-gene expression profile specific for exposure to effective doses of either green tea or polyphenon E. This gene expression signature was altered both in normal lungs and lung adenomas when mice were exposed to green tea or polyphenon E. These experiments identified patterns of gene expressions that both offer clues for green teas potential mechanisms of action and provide a molecular signature specific for green tea exposure.

Effect of an Epidermal Growth Factor Receptor Inhibitor in Mouse Models of Lung Cancer

Gefitinib (Iressa, ZD1839) is a potent high-affinity competitive tyrosine kinase inhibitor aimed primarily at epidermal growth factor receptor (EGFR). Inhibitors in this class have recently been approved for clinical use in the treatment of advanced non–small cell lung cancer as monotherapy following failure of chemotherapy. We examined the efficacy of gefitinib on lung tumorigenesis in mouse models using both postinitiation and progression protocols. Gefitinib was given at a dose of 200 mg/kg body weight (i.g.) beginning either 2 or 12 weeks following carcinogen initiation. In the postinitiation protocol, gefitinib significantly inhibited both tumor multiplicity (∼70%) and tumor load (∼90%) in A/J or p53-mutant mice (P < 0.0001). Interestingly, gefitinib was also highly effective against lung carcinogenesis in the progression protocol when individual animals already have multiple preinvasive lesions in the lung. Gefitinib exhibited ∼60% inhibition of tumor multiplicity and ∼80% inhibition of tumor load when compared with control mice (both P < 0.0001). These data show that gefitinib is a potent chemopreventive agent in both wild-type and p53-mutant mice and that a delayed administration was still highly effective. Analyses of mutations in the EGFR and K-ras genes in lung tumors from either control or treatment groups showed no mutations in EGFR and consistent mutation in K-ras. Using an oligonucleotide array on control and gefitinib-treated lesions showed that gefitinib treatment failed to alter the activity or the expression level of EGFR. In contrast, gefitinib treatment significantly altered the expression of a series of genes involved in cell cycle, cell proliferation, cell transformation, angiogenesis, DNA synthesis, cell migration, immune responses, and apoptosis. Thus, gefitinib showed highly promising chemopreventive and chemotherapeutic activity in this mouse model of lung carcinogenesis. (Mol Cancer Res 2006;4(12):971–81)

Quantitative Receptor-Based Imaging of Tumor Proliferation with the Sigma-2 Ligand ( 18 F)ISO-1

The sigma-2 receptor is expressed in higher density in proliferating (P) tumor cells versus quiescent (Q) tumor cells, thus providing an attractive target for imaging the proliferative status (i.e., P:Q ratio) of solid tumors. Here we evaluate the utility of the sigma-2 receptor ligand 2-(2-[18F]fluoroethoxy)-N-(4-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1H)-yl)butyl)-5-methyl-benzamide, [18F]ISO-1, in two different rodent models of breast cancer. In the first study, small animal Positron Emission Tomography (PET) imaging studies were conducted with [18F]ISO-1 and 18FDG in xenografts of mouse mammary tumor 66 and tracer uptake was correlated with the in vivo P:Q ratio determined by flow cytometric measures of BrdU-labeled tumor cells. The second model utilized a chemically-induced (N-methyl-N-nitrosourea [MNU]) model of rat mammary carcinoma to correlate measures of [18F]ISO-1 and FDG uptake with MR-based volumetric measures of tumor growth. In addition, [18F]ISO-1 and FDG were used to assess the response of MNU-induced tumors to bexarotene and Vorozole therapy. In the mouse mammary 66 tumors, a strong linear correlation was observed between the [18F]ISO-1 tumor: background ratio and the proliferative status (P:Q ratio) of the tumor (R = 0.87). Similarly, measures of [18F]ISO-1 uptake in MNU-induced tumors significantly correlated (R = 0.68, P<0.003) with changes in tumor volume between consecutive MR imaging sessions. Our data suggest that PET studies of [18F]ISO-1 provide a measure of both the proliferative status and tumor growth rate, which would be valuable in designing an appropriate treatment strategy.

A dominant-negative c-jun mutant inhibits lung carcinogenesis in mice.

Lung cancer is the leading cause of cancer mortality in the United States and worldwide. The identification of key regulatory and molecular mechanisms involved in lung tumorigenesis is therefore critical to increase our understanding of this disease and could ultimately lead to targeted therapies to improve prevention and treatment. Induction of members of the activator protein-1 (AP-1) transcription factor family has been described in human non–small cell lung carcinoma. Activation of AP-1 can either stimulate or repress transcription of multiple gene targets, ultimately leading to increased cell proliferation and inhibition of apoptosis. In the present study, we show induction of AP-1 in carcinogen-induced mouse lung tumors compared with surrounding normal lung tissue. We then used a transgenic mouse model directing conditional expression of the dominant-negative c-jun mutant TAM67 in lung epithelial cells to determine the effect of AP-1 inhibition on mouse lung tumorigenesis. Consistent with low AP-1 activity in normal lung tissue, TAM67 expression had no observed effects in adult mouse lung. TAM67 decreased tumor number and overall lung tumor burden in chemically induced mouse lung tumor models. The most significant inhibitory effect was observed on carcinoma burden compared with lower-grade lesions. Our results support the concept that AP-1 is a key regulator of mouse lung tumorigenesis, and identify AP-1–dependent transcription as a potential target to prevent lung tumor progression. Cancer Prev Res; 3(9); 1148–56. ©2010 AACR.

Fine mapping and candidate gene analyses of Pulmonary Adenoma Resistance 1, a major genetic determinant of mouse lung adenoma resistance

Pulmonary adenoma resistance 1 (Par1) is a major genetic determinant of mouse lung adenoma resistance. Although Par1 was previously mapped to mouse chromosome 11 by conventional linkage analyses, its candidate region was broad and undefined. In our present study, we generated Par1 congenic mice using two mouse strains A/J (Par1/-) and Mus spretus (Par1/+). Analyzing these congenic mice enabled us to fine map the Par1 quantitative trait loci (QTL) into a 2.0-cM (2.2 Mb) chromosomal region between genetic marker D11Mit70 and the gene Hoxb9. We then conducted systematic candidate gene screening through nucleotide polymorphism and expression analyses. Genes showing differential lung tissue expression or carrying nonsynonymous single nucleotide polymorphisms were identified and discussed. In particular, we evaluated tumor suppressor gene Tob1 for its Par1 candidacy. Our findings have narrowed the Par1 QTL region and will greatly facilitate the identification of the major genetic determinant of mouse lung adenoma resistance.

Transcriptomic analysis by RNA-seq reveals AP-1 pathway as key regulator that green tea may rely on to inhibit lung tumorigenesis

Green tea is a promising chemopreventive agent for lung cancer. Multiple signaling events have been reported, however, the relative importance of these mechanisms in mediating the chemopreventive function of green tea is unclear. In the present study, to examine the involvement of AP‐1 in green tea polyphenols induced tumor inhibition, human NSCLC cell line H1299 and mouse SPON 10 cells were identified as AP‐1 dependent, as these two lines exhibit high constitutive AP‐1 activity, and when TAM67 expression was induced with doxycycline, cell growth was inhibited and correlated with suppressed AP‐1 activity. RNA‐seq was used to determine the global transcriptional effects of AP‐1 inhibition and also uncover the possible involvement of AP‐1 in tea polyphenols induced chemoprevention. TAM67 mediated changes in gene expression were identified, and within down‐regulated genes, AP‐1 was identified as a key transcription regulator. RNA‐seq analysis revealed that Polyphenon E‐treated cells shared 293 commonly down‐regulated genes within TAM67 expressing H1299 cells, and by analysis of limited Chip‐seq data, over 10% of the down‐regulated genes contain a direct AP‐1 binding site, indicating that Polyphenon E elicits chemopreventive activity by regulating AP‐1 target genes. Conditional TAM67 expressing transgenic mice and NSCLC cell lines were used to further confirm that the chemopreventive activity of green tea is AP‐1 dependent. Polyphenon E lost its chempreventive function both in vitro and in vivo when AP‐1 was inhibited, indicating that AP‐1 inhibition is a major pathway through which green tea exhibits chemopreventive effects.

Enhanced Lung Tumor Development in Tobacco Smoke-Exposed p53 Transgenic and Kras2 Heterozygous Deficient Mice

A/J mice bearing either a mutation in the p53 gene or a Kras2 heterozygous deficiency were investigated for their susceptibility to tobacco smoke-induced lung tumorigenesis. Transgenic mice and their wild-type littermates were exposed to mainstream tobacco smoke (MS) for 5 mo, followed by 4 mo of recovery in filtered air. In sham (filtered air) groups, p53 transgenic mice did not exhibit a higher tumor multiplicity but did exhibit larger tumors, with tumor load increased 3.6-fold, when compared with wild-type mice. With exposure to MS, tumor multiplicity was increased 60% but there was a strikingly increased tumor load (15.9-fold) in p53 transgenic mice. Increased tumor load (5.3-fold) but not tumor multiplicity was seen in MS-exposed Kras2 heterozygous deficient mice. Interestingly, MS exposure did not increase benzo[a]pyrene-induced lung tumorigenesis when MS exposure was initiated after BaP treatment. These results indicate that a p53 mutation or loss of a Kras2 allele increases susceptibility to MS-induced lung tumor development.

Multi-modality imaging of N-methyl-N-nitrosourea-induced mammary tumors by MRI and small animal PET.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #6008 Background: We evaluated the use of high resolution Magnetic Resonance Imaging (MRI) and small animal Positron Emission Tomography (PET) imaging in assessing tumor proliferation and response to therapy in N-methyl-N-nitrosourea (MNU)-induced mammary tumors. Materials and Methods: The study comprised of 30 untreated MNU rats, 6 Targretin-treated MNU rats, and 6 Vorozole-treated MNU rats. Rats received a baseline imaging session when they developed their first palpable mammary tumor and were subsequently randomized into a control group or a treatment group with either Targretin (220mg/kg in the diet) or Vorozole (1.25mg/kg body weight by gavage). Treatment lasted for 8 weeks following the baseline imaging session. Each rat was imaged for 10-weeks at 2-week intervals with a) FDG to assess the metabolic state of tumors, b) MRI to monitor tumor volume, and c) [18F]ISO-1, a Sigma-2 radiolabeled ligand, to assess the proliferative status of MNU-induced tumors. Static images (10-minute) were obtained 60-minutes post-injection of FDG and [18F]ISO-1. In untreated tumors, a minimum of 40 PET outcome measures were assessed for their ability to predict changes in MRI-derived volume measurements between consecutive imaging sessions. In addition, regression analysis was performed to assess the correlation (R) between the measures. In treated tumors, the time-course of changes in PET outcome measures relative to baseline was evaluated to assess the efficacy of Targretin and Vorozole, in particular in characterizing short-term response (initial 2-weeks) and response to treatment withdrawal (weeks 8-10). Results: An index characterizing a MRI-normalized [18F]ISO-1 uptake significantly correlated (R=0.7, P<0.0003) with changes in tumor volume between consecutive imaging sessions in untreated tumors. In contrast, there was no correlation between FDG outcome measures and changes in tumor volume. Targretin had the strongest short-term efficacy with a reduction in tumor load, on average, by as much as 60% compared with Vorozoles 20% reduction in tumor load. We observed higher rate of tumor resurgence in rats treated with Vorozole compared with Targretin, suggesting a residual effect of Targretin. In general, PET outcome measures of both FDG and [18F]ISO-1 delineated short-term response to treatment and non-responding tumors. [18F]ISO-1 was more sensitive than FDG in capturing tumor growth following treatment withdrawal at week 8. Conclusion: MRI and small animal PET provide a platform to assess MNU-induced tumor proliferation and the efficacy of therapeutic interventions non-invasively, as demonstrated in this work. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6008.