Induced Lung Tumors Research Articles

Induction of Lung Tumors and Mutational Analysis in FVB/N Mice Treated with the Tobacco Carcinogen 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone.

Lung cancer remains the leading cause of cancer-related deaths worldwide. In order to understand lung cancer biology and evaluate novel therapeutic strategies, preclinical mouse models have been developed that mimic early and advanced-stage lung cancer. Among autochthonous models, carcinogen-induced systems are valuable preclinical tools since tobacco smoking remains the number one risk factor for lung tumor development. Among the several thousand chemicals within cigarette smoke, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent carcinogen with tumorigenic effects described in both mice and humans. Herein, we describe the methodology for inducing lung tumors in mice using the tobacco carcinogen NNK and subsequent lung fixation for quantitative assessment of tumor development and analysis of oncogenic mutations in tumors.

PRMT6 Promotes Lung Tumor Progression via the Alternate Activation of Tumor-Associated Macrophages.

Increased expression of protein arginine methyl transferase 6 (PRMT6) correlates with worse prognosis in lung cancer cases. To interrogate the in vivo functions of PRMT6 in lung cancer, we developed a tamoxifen-inducible lung-targeted PRMT6 gain-of-function mouse model, which mimics PRMT6 amplification events in human lung tumors. Lung-targeted overexpression of PRMT6 accelerated cell proliferation de novo and potentiated chemical carcinogen (urethane)-induced lung tumor growth. To explore the molecular mechanism/s by which PRMT6 promotes lung tumor growth, we used proteomics-based approaches and identified interleukin-enhancer binding protein 2 (ILF2) as a novel PRMT6-associated protein. Furthermore, by using a series of in vitro gain-of-function and loss-of-function experiments, we defined a new role for the PRMT6-ILF2 signaling axis in alternate activation of tumor-associated macrophages (TAM). Interestingly, we have also identified macrophage migration inhibitory factor, which has recently been shown to regulate alternate activation of TAMs, as an important downstream target of PRMT6-ILF2 signaling. Collectively, our findings reveal a previously unidentified noncatalytic role for PRMT6 in potentiating lung tumor progression via the alternate activation of TAMs. IMPLICATIONS: This is the first study to demonstrate an in vivo role for PRMT6 in lung tumor progression via the alternate activation of TAMs.

Abstract 5189: Activity of polycomb repressive complex 2 determines sensitivity to epigenetic therapy

Abstract EZH2 is a histone methyltransferase that tri-methylates histone H3 at lysine 27 (H3K27me) in the context of the Polycomb Repressive Complex 2, and is highly correlated with poor prognosis of many cancers. However, recent data suggest a de-coupling of EZH2 expression from its enzymatic activity, and many poor prognosis tumors also have low H3K27me3. Therefore, we hypothesize that deletion of Ezh2 would drive more aggressive tumors which may have distinct epigenetic vulnerabilities. To test this hypothesis, we generated the LSL:KrasG12D/+; p53 flox/flox (LSL: lox-stop-lox) mice (Kras/p53 mice) with either zero, one or two floxed alleles of Ezh2 and induced lung tumors with intranasal adeno-Cre virus administration. Supporting our hypothesis, we observed mice with two floxed alleles of Ezh2 (Ezh2 null) had higher tumor burden, higher tumor grade and lower survival than Ezh2 wild-type or heterozygous Kras/p53 mice. Ezh2 null tumors also had a higher propensity to metastasize than Ezh2 wild-type tumors. Consistent with these results, Ezh2 null Kras/p53 tumors and cell lines had increased expression of genes involved in epithelial-mesenchymal transition (EMT), KRAS-driven tumorigenesis, metastasis, and cell cycle regulation. In order to examine the therapeutic vulnerabilities of Ezh2 depleted lung cancers, we built a panel of 2-dimensional cell lines and 3-dimensional organoid cultures of the Kras/p53 tumors that were Ezh2 null, heterozygous and wild-type. We reasoned that using the H3K27me3 demethylase inhibitor might be able to stabilize the residual H3K27me3 in Ezh2 null cells and decrease their aggressive properties. Intriguingly, while we observed no difference in sensitivity to H3K27me3 demethylase inhibitor in the various Ezh2 genotypes of 2-dimensional cultures, both 3-dimensional cultures and in vivo allografts demonstrated Ezh2 null tumors but not Ezh2 WT or heterozygous are sensitive to the H3K27me3 demethylase inhibitor. These data are interesting because they suggest that assays of 3-dimensional organoids may more faithfully recapitulate responses to epigenetic drugs than standard cell lines, and that aggressive tumors with low H3K27me3 might be selectively killed by demethylase inhibitor. We are now expanding our studies to include other targeting agents, including inhibitors of the kinase WEE1 and inhibitor of the chromatin reader BRD4. Citation Format: Fan Chen, Christine F. Brainson. Activity of polycomb repressive complex 2 determines sensitivity to epigenetic therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5189.

Type of TP53 mutation influences oncogenic potential and spectrum of associated K‐ras mutations in lung‐specific transgenic mice

TP53 and K-ras mutations are two of the major genetic alterations in human nonsmall cell lung cancers. The association between these two genes during lung tumorigenesis is unknown. We evaluated the potential of two common Type I (273H, contact) and Type II (175H, conformational) TP53 mutations to induce lung tumors in transgenic mice, as well as K-ras status, and other driver mutations in these tumors. Among 516 (138 nontransgenic, 207 SPC-TP53-273H, 171 SPC-TP53-175H) mice analyzed, 91 tumors, all adenocarcinomas, were observed. Type II mutants developed tumors more frequently (as compared to nontransgenics, p = 0.0003; and Type I, p = 0.010), and had an earlier tumor onset compared to Type I (p = 0.012). K-ras mutations occurred in 21 of 50 (42%) of murine lung tumors sequenced. For both the nontransgenic and the SPC-TP53-273H transgenics, tumor K-ras codon 12-13 mutations occurred after 13 months with a peak incidence at 16-18 months. However, for the SPC-TP53-175H transgenics, K-ras codon 12-13 mutations were observed as early as 6 months, with a peak incidence between the ages of 10-12 months. Codons 12-13 transversion mutations were the predominant changes in the SPC-TP53-175H transgenics, whereas codon 61 transition mutations were more common in the SPC-TP53-273H transgenics. The observation of accelerated tumor onset, early appearance and high frequency of K-ras codon 12-13 mutations in the Type II TP53-175H mice suggests an enhanced oncogenic function of conformational TP53 mutations, and gains in early genetic instability for tumors containing these mutations compared to contact mutations.

NLRP3 deletion inhibits inflammation-driven mouse lung tumorigenesis induced by benzo(a)pyrene and lipopolysaccharide

BackgroundInflammatory micro-environment has been proposed to play a critical role in lung tumorigenesis. NLRP3 is known as an intracellular receptor involving inflammation and has been reported which is increasingly associated with tumor development, but the role in inflammation-driven lung cancer has not been fully clarified. In this study, we investigated whether lipopolysaccharide (LPS)-induced pulmonary inflammation could contribute to lung tumorigenesis induced by benzo(a)pyrene [B(a)p] in C57BL/6J mice and the role of NLRP3 in the pathogenesis.MethodsNLRP3−/− mice and C57BL/6J mice (wide-type, WT) were instilled intratracheally with B(a)p (1 mg/mouse) once a week for 4 times [the week of the last time of B(a)p treatment named Week 0], and mice were then instilled intratracheally with LPS at Week 3, 2.5 μg/mouse, once every three weeks for 5 times. At Week 30, the incidence, number, size and histopathology of lung tumor were analyzed.ResultsMice exposed to B(a)p or B(a)p plus LPS could induce lung tumors, whereas LPS or vehicles treatment could not induce lung tumorigenesis. In WT mice, B(a)p plus LPS exposure significantly increased tumor incidence, mean tumor count and tumor size of visible tumors of lungs compared with B(a)p treatment alone, and NLRP3 deletion inhibited lung tumorigenesis induced by B(a)p or B(a)p plus LPS. Histopathological examination found LPS-induced pulmonary inflammatory changes enhanced lung tumorigenesis induced by B(a)p in WT mice, deletion of NLRP3 improved the inflammatory changes induced by LPS and the number and size of pathological tumor nests induced by B(a)p or B(a)p plus LPS. In addition, we found B(a)p treatment and B(a)p plus LPS treatment predominately induced the development of adenoma.ConclusionLPS enhanced B(a)p-induced lung tumorigenesis in WT and NLRP3−/− mice of C57BL/6J strain, and NLRP3 deletion inhibits lung tumorigenesis induced by B(a)p or B(a)p plus LPS.

Copy Number Analysis of Lung Tumor Progression in Mice

Background & Hypothesis 
 The xeroderma pigmentosum group C (XPC) gene encodes a protein that repairs DNA damage, like that caused by UV light. Its mutation in patients is associated with increased risk of melanoma. In our mouse model, the Xpc gene has a targeted deletion, leading to increased frequency of lung tumors in a mouse strain typically resistant to lung chemical carcinogenesis. We hypothesize that there will be enrichment of specific copy number variations (CNVs) in the genome throughout tumor progression that can distinguish the different stages of carcinogenesis in lung cancer. 
 Project Methods 
 Urethane treatments induced lung tumors in Xpc -/- (ko) mice. Laser capture microscopy was used to isolate tumor, hyperplasia, and normal tissue from lung sections. After DNA extraction, genomic DNA (gDNA) was quantified using Quantifluor assay. Whole genome amplification was completed using Repli-G kit. PCR, copy number analysis, and restriction enzyme digestion were quality control measures for amplified gDNA. Single nucleotide polymorphism (SNP) microarrays will assess CNVs in samples that are successfully amplified. 
 Results 
 DNA was successfully amplified, as confirmed by the Quantifluor assay. PCR, copy number analysis, and restriction enzyme digestion confirmed 3 out of the 4 amplified samples were comparable to matched non-amplified samples, suggesting mouse gDNA was maintained. 
 Potential Impact 
 The mouse model findings can be applied to publically available human genome databases of lung cancer CNVs. Overlapping findings in mice and human CNVs may give insight into novel pathways for effective treatment in lung cancer.

Primary and secondary pyrrolic metabolites of pyrrolizidine alkaloids form DNA adducts in human A549 cells

Humans and animals can be exposed to carcinogenic pyrrolizidine alkaloids (PAs) through consumption of plants commonly found in many parts of the world. Although the liver is the primary target organ for carcinogenic PAs, they have also induced lung tumors in rodents. Hepatic cytochrome P450 activity converts PAs into dehydro-PAs that can be hydrolyzed to dehydropyrrolizidine (DHP); these reactive pyrrolic metabolites can produce four characteristic DNA adducts associated with PA-induced liver tumor initiation in laboratory animals. We reported recently that these four DNA adducts are also formed when 7-glutathione-DHP (7-GS-DHP) or 7-cysteine-DHP is incubated with calf thymus DNA. Here we showed that the four characteristic DNA adducts were formed when human A549 brochoalveolar carcinoma cells were treated with three dehydro-PAs (dehydroriddelliine, dehydromonocrotaline, or dehydroretronecine) or with 7-GS-DHP or 7-cysteine-DHP. For comparison, two parent PAs (riddelliine and monocrotaline) and 7,9-di-glutathionine-DHP were studied. No DHP-DNA adducts were detected with these incubations, confirming that A549 lung carcinoma cells do not express cytochrome P450 enzymes required for metabolic activation of PAs. Our results show that primary and secondary pyrrolic metabolites of carcinogenic PAs produce characteristic DHP-containing DNA adducts in A549 lung cancer cells, suggesting that they are DNA reactive metabolites.

Metabolism of 4-methylimidazole in Fischer 344 rats and B6C3F1 mice

4-Methylimidazole (4-MeI) is a widely used chemical, also identified as a by-product of heating foods. In cancer bioassays, 4-MeI induced lung tumors in mice, but not in rats. To establish if metabolic differences could explain species difference in carcinogenicity, this study investigated metabolism of 4-MeI in rat and mouse lung and liver microsomes and S-9 fractions, and in vivo in rats and mice. No metabolites were detected in rat or mouse lung and liver microsomes, or lung S-9 fractions. Male and female F-344 rats and B6C3F1 mice were administered 50 and 150 mg/kg [14C] 4-MeI by gavage. Excreta, exhaled CO2 and volatiles were collected for 48 h. Elimination was mainly via urine, with 79–89% of the radioactivity in urine in rats and 41–70% in mice. Most of the radioactivity (71–88%) in urine was unchanged 4-MeI. Additional radioactive peaks (the largest metabolite was 8–18%) were characterized by LC-MS/MS as 4-hydroxymethylimidazole, its glucuronide, and other oxidized products, including methylhydantoin. 4-MeI was largely excreted unchanged in rats and mice with limited oxidative metabolism and conjugation. 4-MeI was not oxidized in subcellular fractions from rat and mouse lung and liver. Overall, the metabolism of 4-MeI appeared similar between rats and mice.

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

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

Abstract 5098: Conditional ERK3 overexpression and PTEN deletion induce lung tumors in mice

Abstract Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogen-activated protein kinase (MAPK) involved in cell growth and differentiation. Recent studies have revealed important roles for ERK3 in promoting cancer cell migration and invasion. In addition, ERK3 was shown to be highly upregulated in human lung cancers and to be associated with tumor metastasis. Altogether, these findings suggest an important role for ERK3 in lung tumor formation and progression. However, no in vivo study of ERK3 in lung tumorigenesis has been reported. For this purpose, a transgenic mouse model conditionally overexpressing ERK3 in lung bronchial epithelial cells was generated. First, a transgenic mouse line harboring ERK3 transgene under the control of a ubiquitous promoter and a STOP sequence flanked by two Lox P sites (LSL-ERK3) was generated. The obtained LSL-ERK3 mice were then crossed with a mouse line harboring the Cre recombinase transgene driven by a Clara cell secretory protein gene promoter (CCSP-Cre), resulting in CCSP-Cre/LSL-ERK3 transgenic mice that show ERK3 overexpression in Clara cells, the non-ciliated epithelial cells lining the bronchioles of lung. No clear phenotype, however, was observed in CCSP-Cre/LSL-ERK3 transgenic mice. As lung tumorigenesis usually requires multiple genetic alterations, the CCSP-Cre/LSL-ERK3 transgenic mice were then crossed with a PTENFlox/Flox mouse line in which the exon 5 of the tumor suppressor gene PTEN (Phosphatase and Tensin homolog) is flanked by two Lox P sites. These crossings led to the generation of CCSP-Cre/LSL-ERK3/PTENFlox/Flox mice which display PTEN deletion and ERK3 overexpression in the Clara cells of lung. Thus, four transgenic mouse groups were included in the study and monitored on daily basis: 1) LSL-ERK3; 2) CCSP-Cre/LSL-ERK3; 3) CCSP-Cre/PTENFlox/Flox; 4) CCSP-Cre/LSL-ERK3/PTENFlox/Flox. Tumor growth or other abnormalities in the lungs were analyzed by examining the whole lungs for surface tumors and by histological examination of lung tissue sections. As reported in previous studies, PTEN deletion alone resulted in lung hyperplasia. Interestingly, while ERK3 overexpression alone didn't cause clear phenotype, a combination of ERK3 overexpression with PTEN deletion in CCSP-Cre/LSL-ERK3/ PTENFlox/Flox mice induced lung tumorigenesis as demonstrated by the formation of surface tumors in lungs. Tumor formation in these CCSP-Cre/LSL-ERK3/PTENFlox/Flox mice was confirmed by hematoxylin and eosin (HE) staining of lung sections. Further, immunohistochemical analysis of differential biomarkers suggested that these tumors correspond to lung adenocarcinomas. Taken together, our study demonstrates a promoting role of ERK3 in lung tumor formation in vivo. Citation Format: Sreeram Vallabhaneni, Marion Morel, Ming-Jer Tsai, Francesco J. Demayo, Weiwen Long. Conditional ERK3 overexpression and PTEN deletion induce lung tumors in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5098.

Abstract 2855: Identification of optimal treatment sequence and acquired resistance mechanisms of ALK inhibitors using EML4-ALK transgenic mouse model

Abstract Introduction: EML4-ALK is a distinct molecular entity that is highly sensitive to ALK tyrosine kinase inhibitors (TKIs). While many trials have showed the superiority of ALK TKIs over cytotoxic chemotherapy, the optimal sequencing of ALK TKIs is still obscure. Acquired resistance to ALK TKIs remains a key challenge, and unknown mechanisms of resistance need to be resolved. In this study, we aimed to identify optimal treatment sequence and acquired resistance mechanisms of ALK TKIs using EML4-ALK transgenic mice model that recapitulates human EML4-ALK lung adenocarcinoma. Methods: The tumorigenesis of EML4-ALK transgenic mice was based on Cre-ERT2/Lox system, and intraperitoneal injection with tamoxifen induced lung tumors. When tumor nodules were observed, EML4-ALK transgenic mice were treated with either crizotinib (150mg/kg) followed by ceritinib (75mg/kg) upon progression (N=13) or with ceritinib followed by crizotinib (N=12) upon progression. Tumor response was evaluated weekly using magnetic resonance imaging. Progression-free survival (PFS) was measured to compare the efficacy between two ALK TKIs. For the analysis of acquired resistance mechanism, whole-exome sequencing, RNA sequencing, and targeted sequencing of ALK gene were performed. Results: Head-to-head comparison of crizotinib and ceritinib was performed in the first-line setting. A total of 13 mice were treated with upfront crizotinib, and mice that showed progressive disease (PD) were crossed over to ceritinib (n=9). A total of 12 mice were treated with upfront ceritinib, and mice that showed PD (n=8) were crossed over to crizotinib. Four mice in each group were sacrificed for analysis of resistance mechanism at the time of PD. The PFS of ceritinib was significantly longer than that of crizotinib [24 weeks (95% CI, 21.4-26.5) vs. 8 weeks (95% CI, 6.5-9.0), P &amp;lt; 0.001]. Subsequent treatment with ceritinib following crizotinib resulted in significantly longer PFS than crizotinib following ceritinib [7 weeks (95% CI, 4.1-9.9) vs 3 weeks (95% CI, 2.5-3.5), P &amp;lt; 0.001]. Altogether, treatment with ceritinib followed by crizotinib showed a prolongation of PFS, compared to crizotinib followed by ceritinib (27 weeks vs. 15 weeks, P &amp;lt; 0.001). We noted that ALK-TKI resistant tumors harbored several copy number variations (CNVs), and nonsynonymous oncogenic mutations, but found no previously reported resistant mechanisms including secondary mutations, or CNVs. Wnt signaling related gene set was increased in both crizotinib- and ceritinib-resistant tumors, and Hippo signaling related gene set was increased in ceritinib-resistant tumors on KEGG pathway-based analysis. Conclusion: Our findings suggest that ceritinib is superior to crizotinib when used in the first-line setting. Novel mechanisms of acquired resistance such as increased Wnt and Hippo signaling need further validation. Citation Format: Kyoung-Ho Pyo, Lim Sun Min, Jae Hwan Kim, Ji Min Lee, Ha Ni Jo, Jae Soek Cho, Mi-Ran Yun, Sung Eun Kim, Hye Ryun Kim, Chun-Feng Xin, Tae-Min Kim, Byoung Chul Cho. Identification of optimal treatment sequence and acquired resistance mechanisms of ALK inhibitors using EML4-ALK transgenic mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2855.

Abstract 274: Selective chemopreventive efficacy of 1,4-phenylenebis(methylene)seleno-aspirin (p-XS-Asp) towards lung cancer

Abstract 1,4-Phenylenebis(methylene)selenocyanate (p-XSC) has been shown to inhibit tobacco carcinogen NNK induced lung tumor development in several animal models. This had placed p-XSC on the National Cancer Institute´s (NCI) list of chemopreventive agents for clinical development, but there were systemic toxicity issues. p-XSC metabolizes through the formation of active bis-selenol (p-XSeH) along with the release of poisonous hydrogen cyanide (HCN). We recently developed p-XS-Asp, with a rationale that it would cleave in vivo to release the active p-XSeH and aspirin, thus making the compound less toxic and possibly more potent than p-XSC. Indeed, we previously presented (AACR Annual Meeting 2014) that p-XS-Asp inhibited NNK-induced lung tumorigenesis in A/J mice more effectively than p-XSC, and was also more tolerable. At doses of 15 ppm and 7.5 ppm Se, p-XS-Asp showed a significantly marked decrease in the percentage of lung cancer incidence in vivo with only 50% and 87% of tumor incidence, as compared to p-XSC (79% and 100%), respectively. NNK-control showed an 100% tumor incidence. Likewise, the tumor multiplicity for p-XS-Asp group was 0.87 and 1.93 tumors/mouse as compared to the NNK-control (11.53) and p-XSC (1.66 and 4.10 tumors/mouse, respectively) at the two doses tested. Notably, blood chemistry and tissue analyses did not show systemic toxicity for the p-XS-Asp fed group. We have now evaluated the underlying mechanisms of lung cancer preventive action of p-XS-Asp and its efficacy for inhibiting azoxymethane (AOM)- and dimethyl hydrazine (DMH)-induced Aberrant Crypt Foci (ACF) in Fischer F344 rats. At a dose of 7.5 ppm Se, p-XS-Asp was able to restore the expression of several genes (MMP9, COX-2, Myc, SphK1 and RELA), that were over-expressed in the NNK group, to control or even lower levels. The AKT1 gene expression was much lower in the lung tissue of p-XS-Asp treated mice at this dose compared to both negative and NNK control groups. Therefore, p-XS-Asp might be exerting its chemopreventive effect on NNK-induce lung tumorigenesis via inhibiting COX-2 mediated PI3K/Akt signaling pathway. Interestingly, contrary to the striking inhibition of lung tumorigenesis, p-XS-Asp failed to significantly inhibit AOM- or DMH-induced formation of ACF in Fischer F344 rats. In both AOM and DMH models, aspirin (positive control) significantly reduced the number of ACF and large ACF per area (cm2) by 37.4% and 33.8%, respectively. On the other hand, both p-XSC and p-XS-Asp showed no significant inhibitory effect on the formation of ACF and large ACF. Taken together, our results have shown p-XS-Asp to selectively prevent the lung, but not colon, tumorigenesis, and thus is a promising candidate for further development as a lung cancer preventive agent. Citation Format: Daniel Plano, Srinivasa Ramisetti, Sang-Yub Kim, Manoj Pandey, Cesar Aliaga, Deepkamal N. Karelia, Arthur Berg, Carmen Sanmartin, Junxuan Lu, Shantu Amin, Arun K. Sharma. Selective chemopreventive efficacy of 1,4-phenylenebis(methylene)seleno-aspirin (p-XS-Asp) towards lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 274.

Abstract 4051: Different immune signatures in advanced lung tumors of Nrf2 wildtype vs. knockout mice

Abstract Activation of Nrf2 and its downstream target genes serves as a master defense mechanism, protecting cells against oxidative and electrophilic stress. Numerous activators of Nrf2 inhibit carcinogenesis. However, genetic alterations resulting in constitutive Nrf2 activation in human lung tumors promotes tumor growth and chemoresistance. The dual role of Nrf2 in cancer has generated a high level of interest, but little is known regarding the effect of Nrf2 on immune cells during lung carcinogenesis. In this study, we used the potent carcinogen vinyl carbamate to induce lung tumors in mice. Tumor burden, the immunophenotype, and genetic signatures were compared in the lungs of Nrf2 knockout (-/-, KO) vs. wildtype (+/+, WT) mice. Nrf2 KO mice were more susceptible to vinyl carbamate; these mice developed tumors earlier than the WT mice and exhibited more and larger tumors over times ranging from 20-32 weeks after initiation. By 32 weeks, WT mice have an average of 6.5 ± 0.95 tumors per lung compared to 12.25 ± 2.6 tumors in the KO mice. Immune cell populations including T cells (CD4+ and CD8+), B cells, macrophages and myeloid derived suppressor cells were investigated in the lungs and spleens of Nrf2 WT and KO mice challenged with vinyl carbamate. T cell populations were significantly (P &amp;lt; 0.05) reduced in the lungs of Nrf2 KO mice, while tumor-promoting macrophages and myeloid derived suppressor cells were elevated in the lung and spleen, respectively, of Nrf2 KO mice compared to WT mice. Moreover, immune response genes, especially a series of cytokines (Cxcl1, Cxcl12, Csf1, and Ccl9) and MHC antigens that promote tumor growth, were significantly upregulated in tumors from Nrf2-/- mice. Importantly, this expression profile is conserved in patients with lung cancer. Overall, our results confirmed a tumor protective role of Nrf2 in carcinogenesis and suggest an important regulatory role for Nrf2 in immune cells in both mouse and human lung tumors. Citation Format: Di Zhang, Jonathan Rennhack, Eran Andrechek, Cheryl Rockwell, Karen Liby. Different immune signatures in advanced lung tumors of Nrf2 wildtype vs. knockout mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4051.

Nrf2-activated expression of sulfiredoxin contributes to urethane-induced lung tumorigenesis

Lung cancer is the leading cause of cancer death worldwide. Cigarette smoking and exposure to chemical carcinogens are among the risk factors of lung tumorigenesis. In this study, we found that cigarette smoke condensate and urethane significantly stimulated the expression of sulfiredoxin (Srx) at the transcript and protein levels in cultured normal lung epithelial cells, and such stimulation was mediated through the activation of nuclear related factor 2 (Nrf2). To study the role of Srx in lung cancer development in vivo, mice with Srx wildtype, heterozygous or knockout genotype were subjected to the same protocol of urethane treatment to induce lung tumors. By comparing tumor multiplicity and volume between groups of mice with different genotype, we found that Srx knockout mice had a significantly lower number and smaller size of lung tumors. Mechanistically, we demonstrated that loss of Srx led to a decrease of tumor cell proliferation as well as an increase of tumor cell apoptosis. These data suggest that Srx may have an oncogenic role that contributes to the development of lung cancer in smokers or urethane-exposed human subjects.

Abstract A22: Ezh2 haploinsufficiency and full insufficiency in KRAS/p53-null lung tumors drive distinct cellular phenotypes

Abstract EZH2, the functional enzymatic component of the Polycomb Repressive Complex 2 (PRC2), is a histone methyltransferase that trimethylates histone H3 at lysine 27 (H3K27me), and may act as an oncogene or tumor suppressor in different cellular contexts. EZH2 overexpression correlates with worse prognosis in many cancer types including breast, lung, and prostate cancer. However, other data suggest EZH2 activation results in better outcomes in colon cancer, and several publications show that inactivating mutations in EZH2 and its related proteins are common in T-cell acute lymphoblastic leukemia (T-ALL). Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. Because EZH2 inhibitors are currently in clinical trials for many cancer types, understanding the roles of EZH2 in lung tumor initiation and progression is important. We generated the LSL:KrasG12D/+; p53 flox/flox (LSL: lox-stop-lox) mice (Kras/p53 mice) with either zero, one or two floxed alleles of Ezh2 and induced lung tumors with intranasal adeno-Cre virus administration. Similar to findings by other groups, mice with two floxed alleles of Ezh2 (Ezh2 null) had higher tumor burden (57%), higher tumor grade, and lower survival (110 days) than Ezh2 wild-type Kras/p53 mice (45% and 116 days, respectively). Ezh2 null tumors also had a higher propensity to metastasize than Ezh2 wild-type tumors at time-points greater than 110 days after adeno-Cre virus administration (75% vs. 16%, respectively). However, we also observed that mice with only one floxed allele of Ezh2 (Ezh2 heterozygous) had lower tumor burden (27%) and grade and increased survival (127 days) compared to Ezh2 wild-type mice. These data suggest that full and partial insufficiency for Ezh2 have distinct phenotypic outcomes. We generated two-dimensional tumor cell lines, from which the preliminary RNA-seq showed that 108 genes were significantly upregulated and 45 genes significantly downregulated in Ezh2flox/flox cells relative to Ezh2+/+ Kras/p53 cells (FDR&amp;lt;0.05, LFC&amp;gt;1.3). GSEA results of Ezh2 null vs. wild-type positively correlated with epithelial-mesenchymal transition (EMT), KRAS-driven tumorigenesis, metastasis, Hippo signaling, and imprinted genes. In contrast, Ezh2 heterozygous vs. wild-type showed enrichment of non-metastasis signatures and only partial enrichment of PRC2-target and imprinted genes. Consistent with RNA-seq results, Ezh2 null cell lines showed obvious EMT morphology compared to Ezh2 wild-type cells. ChIP-seq studies to learn how H3K27me3 correlates with gene expression are ongoing. In conclusion, Ezh2 haploinsufficiency (heterozygous) leads to better prognosis while full insufficiency (null) causes aggressive metastatic lung tumors in the Kras/p53 mouse model. Given that loss of the H3K27me3 methyltransferase Ezh2 leads to more aggressive KRAS-driven tumors, we are now exploring the efficacy of the H3K27me3 demethylase inhibitors in this genotype. Three-dimensional cultures of murine tumor cells have demonstrated sensitivity to the H3K27me3 demethylase inhibitors GSK-J4, which may be a method to epigenetically target KRAS+ tumors. Citation Format: Fan Chen, Aria L. Byrd, Yanming Zhao, Carla F. Kim, Christine Fillmore Brainson. Ezh2 haploinsufficiency and full insufficiency in KRAS/p53-null lung tumors drive distinct cellular phenotypes [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A22.

Germline control of somatic Kras mutations in mouse lung tumors.

Somatic KRAS mutations are common in human lung adenocarcinomas and are associated with worse prognosis. In mice, Kras is frequently mutated in both spontaneous and experimentally induced lung tumors, although the pattern of mutation varies among strains, suggesting that such mutations are not random events. We tested if the occurrence of Kras mutations is under genetic control in two mouse intercrosses. Codon 61 mutations were prevalent, but the patterns of nucleotide changes differed between the intercrosses. Whole genome analysis with SNPs in (A/J x C57BL/6)F4 mice revealed a significant linkage between a locus on chromosome 19 and 2 particular codon 61 variants (CTA and CGA). In (AIRmax × AIRmin) F2 mice, there was a significant linkage between SNPs located on distal chromosome 6 (around 135 Mbp) and the frequency of codon 61 mutation. These results reveal the presence of two loci, on chromosomes 6 and 19, that modulate Kras mutation frequency in different mouse intercrosses. These findings indicate that somatic mutation frequency and type are not simple random events, but are under genetic control.

IL-32 gamma reduces lung tumor development through upregulation of TIMP-3 overexpression and hypomethylation

The low expression of tissue inhibitor of metalloproteinase 3 (TIMP-3) is important in inflammatory responses. Therefore, inhibition of TIMP-3 may promote tumor development. Our study showed that expression of TIMP-3 was elevated in lL-32γ mice lung tissues. In this study, we investigated whether IL-32γ mice inhibited lung tumor development through overexpression of TIMP-3 and its methylation. To explore the possible underlying mechanism, lung cancer cells were transfected with IL-32γ cDNA plasmid. A marked increase in TIMP-3 expression was caused by promoter methylation. Mechanistic studies indicated that TIMP-3 overexpression reduced NF-κB activity, which led to cell growth inhibition in IL-32γ transfected lung cancer cells. We also showed that IL-32γ inhibits expression of DNA (cytosine-5-)-methyltransferase 1 (DNMT1). Moreover, IL-32γ inhibits the binding of DNMT1 to TIMP-3 promoter, but this effect was reversed by the treatment of DNA methyltransferase inhibitor (5-Aza-CdR) and NF-κB inhibitor (PS1145), suggesting that a marked increase in TIMP-3 expression was caused by inhibition of promoter hypermethylation via decreased DNMT1 expression through the NF-κB pathway. In an in vivo carcinogen induced lung tumor model, tumor growth was inhibited in IL-32γ overexpressed mice with elevated TIMP-3 expression and hypomethylation accompanied with reduced NF-κB activity. Moreover, in the lung cancer patient tissue, the expression of IL-32 and TIMP-3 was dramatically decreased at a grade-dependent manner compared to normal lung tissue. In summary, IL-32γ may increase TIMP-3 expression via hypomethylation through inactivation of NF-κB activity, and thereby reduce lung tumor growth.

Noncanonical agonist PPARγ ligands modulate the response to DNA damage and sensitize cancer cells to cytotoxic chemotherapy

The peroxisome-proliferator receptor-γ (PPARγ) is expressed in multiple cancer types. Recently, our group has shown that PPARγ is phosphorylated on serine 273 (S273), which selectively modulates the transcriptional program controlled by this protein. PPARγ ligands, including thiazolidinediones (TZDs), block S273 phosphorylation. This activity is chemically separable from the canonical activation of the receptor by agonist ligands and, importantly, these noncanonical agonist ligands do not cause some of the known side effects of TZDs. Here, we show that phosphorylation of S273 of PPARγ occurs in cancer cells on exposure to DNA damaging agents. Blocking this phosphorylation genetically or pharmacologically increases accumulation of DNA damage, resulting in apoptotic cell death. A genetic signature of PPARγ phosphorylation is associated with worse outcomes in response to chemotherapy in human patients. Noncanonical agonist ligands sensitize lung cancer xenografts and genetically induced lung tumors to carboplatin therapy. Moreover, inhibition of this phosphorylation results in deregulation of p53 signaling, and biochemical studies show that PPARγ physically interacts with p53 in a manner dependent on S273 phosphorylation. These data implicate a role for PPARγ in modifying the p53 response to cytotoxic therapy, which can be modulated for therapeutic gain using these compounds.

Modeling Respiratory Induced Lung Tumor Motion for Highly Efficient Radiotherapy

Modeling Respiratory Induced Lung Tumor Motion for Highly Efficient Radiotherapy

The “false-positive” conundrum in the NTP 2-year rodent cancer study database

In 1990, Ames and Gold described a conundrum of “too many carcinogens” among chemicals tested in rodent bioassays. Their proposed nongenotoxic carcinogenic mechanism was amplification of the background mutation rate via cytotoxicity induced by high doses of the test chemicals, thereby leading to increases in reparative cellular proliferation rates. Recently, we have statistically and mechanistically analyzed the entire 594-study (470 final reports) NTP 2-year rodent cancer database to better understand the conundrum posed by Ames and Gold. Our analysis provides several lines of evidence that support the contention of Ames and Gold. First, across different routes of administration, relatively phylogenetically similar rats and mice are nonetheless discordant for the development of tumors at similar organ sites. Tumor site concordance across sex within species is higher than tumor site concordance across species. Second, many chemicals negative in the Ames test nonetheless induce tumors in either rats or mice. Third, 11 out of 58 chemicals tested by the inhalation route induce lung tumors in mice and not rats, are negative in the Ames test, and exhibit hyperplasia. In 2017, Tomasetti et al. provided evidence for the clinical relevance in humans of the Ames and Gold mechanism regarding amplification of the background mutation rate by demonstrating that the majority of human tumors result from accumulated mutations due to DNA replication errors.