M. Gerard O'Sullivan

A transposon-based genetic screen in mice identifies genes altered in colorectal cancer

Human colorectal cancers (CRCs) display a large number of genetic and epigenetic alterations, some of which are causally involved in tumorigenesis (drivers) and others that have little functional impact (passengers). To help distinguish between these two classes of alterations, we used a transposon-based genetic screen in mice to identify candidate genes for CRC. Mice harboring mutagenic Sleeping Beauty (SB) transposons were crossed with mice expressing SB transposase in gastrointestinal tract epithelium. Most of the offspring developed intestinal lesions, including intraepithelial neoplasia, adenomas, and adenocarcinomas. Analysis of over 16,000 transposon insertions identified 77 candidate CRC genes, 60 of which are mutated and/or dysregulated in human CRC and thus are most likely to drive tumorigenesis. These genes include APC, PTEN, and SMAD4. The screen also identified 17 candidate genes that had not previously been implicated in CRC, including POLI, PTPRK, and RSPO2.

A Sleeping Beauty transposon-mediated screen identifies murine susceptibility genes for adenomatous polyposis coli (Apc)-dependent intestinal tumorigenesis

It is proposed that a progressive series of mutations and epigenetic events leads to human colorectal cancer (CRC) and metastasis. Furthermore, data from resequencing of the coding regions of human CRC suggests that a relatively large number of mutations occur in individual human CRC, most at low frequency. The functional role of these low-frequency mutations in CRC, and specifically how they may cooperate with high-frequency mutations, is not well understood. One of the most common rate-limiting mutations in human CRC occurs in the adenomatous polyposis coli (APC) gene. To identify mutations that cooperate with mutant APC, we performed a forward genetic screen in mice carrying a mutant allele of Apc (ApcMin) using Sleeping Beauty (SB) transposon-mediated mutagenesis. ApcMin SB-mutagenized mice developed three times as many polyps as mice with the ApcMin allele alone. Analysis of transposon common insertion sites (CIS) identified the Apc locus as a major target of SB-induced mutagenesis, suggesting that SB insertions provide an efficient route to biallelic Apc inactivation. We also identified an additional 32 CIS genes/loci that may represent modifiers of the ApcMin phenotype. Five CIS genes tested for their role in proliferation caused a significant change in cell viability when message levels were reduced in human CRC cells. These findings demonstrate the utility of using transposon mutagenesis to identify low-frequency and cooperating cancer genes; this approach will aid in the development of combinatorial therapies targeting this deadly disease.

Inhaled carbon monoxide reduces leukocytosis in a murine model of sickle cell disease

Carbon monoxide (CO) has anti-inflammatory properties. We previously reported that acute treatments with inhaled CO inhibit vascular inflammation and hypoxia-induced vasoocclusion in sickle cell disease mouse models. Therefore, we hypothesized that chronic CO inhalation would decrease vascular inflammation and organ pathology in a sickle cell disease mouse model. The treatment of sickle cell disease mice with 25 or 250 parts/million inhaled CO for 1 h/day, 3 days/wk for 8-10 wk significantly decreased the total mean white blood cell, neutrophil, and lymphocyte counts in peripheral blood. Eight weeks of 250 parts/million CO treatments reduced staining for myeloid and lymphoid markers in the bone marrow of sickle mice. Bone marrow from treated sickle mice exhibited a significant decrease in colony-forming unit granulocyte-macrophage during colony-forming cell assays. Anti-inflammatory signaling pathways phospho-Akt and phospho-p38 MAPK were markedly increased in CO-treated sickle livers. Importantly, CO-treated sickle mice had a significant reduction in liver parenchymal necrosis, reflecting the anti-inflammatory benefits of CO. We conclude that inhaled CO may be a beneficial anti-inflammatory therapy for sickle cell disease.

Chemopreventive Effect of Kava on 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone plus Benzo[a]pyrene–Induced Lung Tumorigenesis in A/J Mice

Lung cancer is the leading cause of cancer death, and chemoprevention is a potential strategy to help control this disease. Epidemiologic survey indicates that kava may be chemopreventive for lung cancer, but there is a concern about its potential hepatotoxicity. In this study, we evaluated whether oral kava could prevent 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) plus benzo[a]pyrene (B[a]P)–induced lung tumorigenesis in A/J mice. We also studied the effect of kava to liver. At a dose of 10 mg/g diet, 30-week kava treatment (8 weeks concurrent with NNK and B[a]P treatment followed by 22 weeks post-carcinogen treatment) effectively reduced lung tumor multiplicity by 56%. Kava also reduced lung tumor multiplicity by 47% when administered concurrently with NNK and B[a]P for 8 weeks. Perhaps most importantly, kava reduced lung tumor multiplicity by 49% when administered after the final NNK and B[a]P treatment. These results show for the first time the chemopreventive potential of kava against lung tumorigenesis. Mechanistically, kava inhibited proliferation and enhanced apoptosis in lung tumors, as shown by a reduction in proliferating cell nuclear antigen (PCNA), an increase in caspase-3, and cleavage of poly(ADP-ribose) polymerase (PARP). Kava treatment also inhibited the activation of nuclear factor κBNF-κB, a potential upstream mechanism of kava chemoprevention. Although not rigorously evaluated in this study, our preliminary data were not suggestive of hepatotoxicity. Based on these results, further studies are warranted to explore the chemopreventive potential and safety of kava.

Vaccination for Invasive Canine Meningioma Induces in Situ Production of Antibodies Capable of Antibody-Dependent Cell-Mediated Cytotoxicity

Malignant and atypical meningiomas are resistant to standard therapies and associated with poor prognosis. Despite progress in the treatment of other tumors with therapeutic vaccines, this approach has not been tested preclinically or clinically in these tumors. Spontaneous canine meningioma is a clinically meaningful but underutilized model for preclinical testing of novel strategies for aggressive human meningioma. We treated 11 meningioma-bearing dogs with surgery and vaccine immunotherapy consisting of autologous tumor cell lysate combined with toll-like receptor ligands. Therapy was well tolerated, and only one dog had tumor growth that required intervention, with a mean follow up of 585 days. IFN-γ-elaborating T cells were detected in the peripheral blood of 2 cases, but vaccine-induced tumor-reactive antibody responses developed in all dogs. Antibody responses were polyclonal, recognizing both intracellular and cell surface antigens, and HSP60 was identified as one common antigen. Tumor-reactive antibodies bound allogeneic canine and human meningiomas, showing common antigens across breed and species. Histologic analysis revealed robust infiltration of antibody-secreting plasma cells into the brain around the tumor in posttreatment compared with pretreatment samples. Tumor-reactive antibodies were capable of inducing antibody-dependent cell-mediated cytotoxicity to autologous and allogeneic tumor cells. These data show the feasibility and immunologic efficacy of vaccine immunotherapy for a large animal model of human meningioma and warrant further development toward human trials.

An in vivo immunotherapy screen of costimulatory molecules identifies Fc-OX40L as a potent reagent for the treatment of established murine gliomas

Purpose: We tested the combination of a tumor lysate vaccine with a panel of costimulatory molecules to identify an immunotherapeutic approach capable of curing established murine gliomas. Experimental Design: Glioma-bearing mice were primed with a tumor lysate vaccine, followed by systemic administration of the following costimulatory ligands: OX40L, CD80, 4-1BBL, and GITRL, which were fused to the Fc portion of human immunoglobulin. Lymphocytes and mRNA were purified from the brain tumor site for immune monitoring studies. Numerous variations of the vaccine and Fc-OX40L regimen were tested alone or in combination with temozolomide. Results: Lysate vaccinations combined with Fc-OX40L led to the best overall survival, yielding cure rates of 50% to 100% depending on the timing, regimen, and combination with temozolomide. Cured mice that were rechallenged with glioma cells rejected the challenge, showing immunologic memory. Lymphocytes isolated from the draining lymph nodes of vaccine/Fc-OX40L–treated mice had superior tumoricidal function relative to all other groups. Vaccine/Fc-OX40L–treated mice exhibited a significant increase in proliferation of brain-infiltrating CD4 and CD8 T cells, as indicated by Ki67 staining. Fc-OX40L had single-agent activity in transplanted and spontaneous glioma models, and the pattern of inflammatory gene expression in the tumor predicted the degree of therapeutic response. Conclusions: These data show that Fc-OX40L has unique and potent activity against experimental gliomas and warrants further testing. Clin Cancer Res; 18(17); 4657–68. ©2012 AACR.

Transposon Mutagenesis Screen Identifies Potential Lung Cancer Drivers and CUL3 as a Tumor Suppressor

Non–small cell lung cancers (NSCLC) harbor thousands of passenger events that hide genetic drivers. Even highly recurrent events in NSCLC, such as mutations in PTEN, EGFR, KRAS, and ALK, are detected, at most, in only 30% of patients. Thus, many unidentified low-penetrant events are causing a significant portion of lung cancers. To detect low-penetrance drivers of NSCLC, a forward genetic screen was performed in mice using the Sleeping Beauty (SB) DNA transposon as a random mutagen to generate lung tumors in a Pten-deficient background. SB mutations coupled with Pten deficiency were sufficient to produce lung tumors in 29% of mice. Pten deficiency alone, without SB mutations, resulted in lung tumors in 11% of mice, whereas the rate in control mice was approximately 3%. In addition, thyroid cancer and other carcinomas, as well as the presence of bronchiolar and alveolar epithelialization, in mice deficient for Pten were also identified. Analysis of common transposon insertion sites identified 76 candidate cancer driver genes. These genes are frequently dysregulated in human lung cancers and implicate several signaling pathways. Cullin3 (Cul3), a member of a ubiquitin ligase complex that plays a role in the oxidative stress response pathway, was identified in the screen and evidence demonstrates that Cul3 functions as a tumor suppressor. Implications: This study identifies many novel candidate genetic drivers of lung cancer and demonstrates that CUL3 acts as a tumor suppressor by regulating oxidative stress. Mol Cancer Res; 13(8); 1238–47. ©2015 AACR.

Loss of EphA2 receptor tyrosine kinase reduces ApcMin/+ tumorigenesis.

The Eph receptor A2 (EphA2) is overexpressed in a range of human epithelial cancers, a phenotype that is associated with cancer cell proliferation, progression and angiogenesis. Mouse models of mammary neoplasia have confirmed the role of EphA2 as mice carrying a knockout allele of EphA2 were resistant to breast cancer, a phenotype that was associated with interactions between EphA2 and ErbB2. We investigated in vivo the role of EphA2 in GI cancer. To determine whether EphA2 influences intestinal tumorigenesis, we used qRT‐PCR to examine the mRNA expression levels of EphA2 in tumors from the small intestine and colon of ApcMin/+ mice. We found that EphA2 was significantly up‐regulated in tumors from both regions when compared with normal control tissues. We then evaluated the spatial expression patterns of EphA2 protein using immunohistochemistry in both the small intestine and colon and found that in normal tissues EphA2 was robustly expressed in highly differentiated cells, such as cells of the villi, but that EphA2 expression was largely absent from the stem cell niche and proliferative zones of intestinal crypts. In contrast, in tumors EphA2 was broadly expressed. Finally, we created a strain of ApcMin/+ mice carrying a genetic knockout of the EphA2 gene. These mice developed significantly fewer and smaller tumors in both the small and large intestine. Overall, our results indicate that EphA2 plays an oncogenic role in the mammalian intestine suggesting that strategies to target EphA2 activity may offer new therapeutic modalities for colorectal cancer.

A concomitant loss of dormant origins and FANCC exacerbates genome instability by impairing DNA replication fork progression

Abstract Accumulating evidence suggests that dormant DNA replication origins play an important role in the recovery of stalled forks. However, their functional interactions with other fork recovery mechanisms have not been tested. We previously reported intrinsic activation of the Fanconi anemia (FA) pathway in a tumor-prone mouse model (Mcm4chaos3) with a 60% loss of dormant origins. To understand this further, we introduced a null allele of Fancc (Fancc−), encoding a member of the FA core complex, into the Mcm4chaos3 background. Primary embryonic fibroblasts double homozygous for Mcm4chaos3 and Fancc− (Mcm4chaos3/chaos3;Fancc−/−) showed significantly increased levels of markers of stalled/collapsed forks compared to either single homozygote. Interestingly, a loss of dormant origins also increased the number of sites in which replication was delayed until prophase, regardless of FA pathway activation. These replication defects coincided with substantially elevated levels of genome instability in Mcm4chaos3/chaos3;Fancc−/− cells, resulting in a high rate of perinatal lethality of Mcm4chaos3/chaos3;Fancc−/− mice and the accelerated tumorigenesis of surviving mice. Together, these findings uncover a specialized role of dormant origins in replication completion while also identifying important functional overlaps between dormant origins and the FA pathway in maintaining fork progression, genome stability, normal development and tumor suppression.

Mutagenicity of furan in female Big Blue B6C3F1 mice.

Furan is an abundant food and environmental contaminant that is a potent liver carcinogen in rodent models. To determine if furan is genotoxic in vivo, female B6C3F1 Big Blue transgenic mice were treated with 15 mg/kg bw furan by gavage 5 days a week for 6 weeks, or once weekly for 3 weeks. Liver cII transgene mutation-frequency and mutation spectra were determined. Furan did not increase the mutation frequency under either treatment condition. In the 6-week treatment regimen, there was a change in the cII transgene mutation-spectrum, with the fraction of GC to AT transitions significantly reduced. The only other significant change was an increase in GC to CG transversions; these represented a minor contribution to the overall mutation spectrum. A much larger furan-dependent shift was observed in the 3-week study. There was a significant increase in transversion mutations, predominantly GC to TA transversions as well as smaller non-significant changes in GC to CG and AT to TA transversions. To determine if these mutations were caused by cis-2-butene-1,4-dial (BDA), a reactive metabolite of furan, the mutagenic activity and the mutation spectrum of BDA was determined in vitro, in Big Blue mouse embryonic fibroblasts. This compound did not increase the cII gene mutation-frequency but caused a substantial increase in AT to CG transversions. This increase, however, lost statistical significance when adjusted for multiple comparisons. Together, these findings suggest that BDA may not be directly responsible for the in-vivo effects of furan on mutational spectra. Histopathological analysis of livers from furan-treated mice revealed that furan induced multifocal, hepatocellular necrosis admixed with reactive leukocytes and pigment-laden Kupffer cells, enhanced oval-cell hyperplasia, and increased hepatocyte mitoses, some of which were atypical. An indirect mechanism of genotoxicity is proposed in which chronic toxicity followed by inflammation and secondary cell proliferation triggers cancer development in furan-exposed rodents.