Laura W. Fornwald

Inactivation of ErbB3 by siRNA promotes apoptosis and attenuates growth and invasiveness of human lung adenocarcinoma cell line A549

The ErbB3 receptor and the downstream signaling kinase Akt are implicated in proliferation of lung adenocarcinoma cells. Inhibition by siRNAs to ErbB3 and Akt isoforms 1, 2 and 3 was utilized to investigate the contribution of these molecules to tumor survival, spreading and invasiveness, and the roles of specific Akt isoforms. ErbB3 siRNA stably and dose-dependently suppressed ErbB3 protein for 2 days or more, and reduced cell numbers, by both suppressing cell cycle and causing apoptosis and necrosis. It also inhibited soft agar growth, cell motility and migration, and invasiveness. Akt1, 2 and 3 siRNAs had similar suppressive effects on cell number, apoptosis/necrosis and soft agar growth. However, although Akt1 siRNA had no effect on cell migration or invasion, Akt2 siRNA effectively suppressed both activities, and Akt3 siRNA had moderate effectiveness. In A549 cells, ErbB3 is indicated as having major effects on cell division, survival, motility, migration and invasiveness. All three Akt isoforms are to varying degrees involved in these cell behaviors, with Akt2 especially implicated in migration and invasion. ErbB3 and the Akts are promising targets for therapy, and siRNAs may be useful for this purpose.

K-Ras p21 EXPRESSION AND ACTIVITY IN LUNG AND LUNG TUMORS

Although K-ras is mutated in many human and mouse lung adenocarcinomas, the function of K-ras p21 in lung is not known. We sought evidence for the prevalent hypothesis that K-ras p21 activates raf, which in turn passes the signal through the extracellular signal regulated kinases (Erks) to stimulate cell division, and that this pathway is upregulated when K-ras is mutated. Results from both mouse lung tumors and immortalized cultured E10 and C10 lung type II cells failed to substantiate this hypothesis. Lung tumors did not have more total K-ras p21 or K-ras p21 GTP than normal lung tissue, nor were high levels of these proteins found in tumors with mutant K-ras. Activated K-ras p21-GTP levels did not correlate with proliferating cell nuclear antigen. Special features of tumors with mutant K-ras included small size of carcinomas compared with carcinomas lacking this mutation, and correlation of proliferating cell nuclear antigen with raf-1. In nontransformed type II cells in culture, both total and activated K-ras p21 increased markedly at confluence but not after serum stimulation, whereas both Erk1/2 and the protein kinase Akt were rapidly activated by the serum treatment. Reverse transcriptase-polymerase chain reaction (RT-PCR) assays of K-ras mRNA indicated an increase in confluent and especially in postconfluent cells. Together the findings indicate that normal K-ras p21 activity is associated with growth arrest of lung type II cells, and that the exact contribution of mutated K-ras p21 to tumor development remains to be discovered.

Ontogeny-Driven rDNA Rearrangement, Methylation, and Transcription, and Paternal Influence

Gene rearrangement occurs during development in some cell types and this genome dynamics is modulated by intrinsic and extrinsic factors, including growth stimulants and nutrients. This raises a possibility that such structural change in the genome and its subsequent epigenetic modifications may also take place during mammalian ontogeny, a process undergoing finely orchestrated cell division and differentiation. We tested this hypothesis by comparing single nucleotide polymorphism-defined haplotype frequencies and DNA methylation of the rDNA multicopy gene between two mouse ontogenic stages and among three adult tissues of individual mice. Possible influences to the genetic and epigenetic dynamics by paternal exposures were also examined for Cr(III) and acid saline extrinsic factors. Variables derived from litters, individuals, and duplicate assays in large mouse populations were examined using linear mixed-effects model. We report here that active rDNA rearrangement, represented by changes of haplotype frequencies, arises during ontogenic progression from day 8 embryos to 6-week adult mice as well as in different tissue lineages and is modifiable by paternal exposures. The rDNA methylation levels were also altered in concordance with this ontogenic progression and were associated with rDNA haplotypes. Sperm showed highest level of methylation, followed by lungs and livers, and preferentially selected haplotypes that are positively associated with methylation. Livers, maintaining lower levels of rDNA methylation compared with lungs, expressed more rRNA transcript. In vitro transcription demonstrated haplotype-dependent rRNA expression. Thus, the genome is also dynamic during mammalian ontogeny and its rearrangement may trigger epigenetic changes and subsequent transcriptional controls, that are further influenced by paternal exposures.

Mechanism-based inhibition of mouse P4502b-10 by selected arylalkynes

Suicide inhibitors of cytochrome P450 families are excellent tools to predict which isoforms mediate the metabolism/activation of a variety of chemical agents. We compared the inhibitory effects of several arylalkynes on mouse cytochromes P450 with published data for the rat model. The inhibition of P4502b specific dealkylation of benzyloxyresorufin by 2-ethynylnaphthalene (2-EN), 5-phenyl-1-pentyne (PPY), 4-phenyl-1-butyne (PBY), and 9-ethynylphenanthrene (9-EPh) was measured in hepatic microsomes from male mice treated with 1,4-bis[2-(3,5-dichloropyridyloxy)]-benzene (TCPOBOP) to induce cytochrome P4502b. Pulmonary microsomes were prepared from untreated mice. 9-EPh, 2-EN, and PPY caused a time-, concentration-, and NADPH-dependent loss in P4502b activity in both tissues. PBY, however, demonstrated this type of inhibition only in liver microsomes. The IC50 was calculated for both liver and lung microsomes and compared with published Ki (concentration required for half-maximal inhibition) or KI (concentration required for half-maximal inactivation) values for the rat. PPY, PBY, and 9-EPh were equally effective inhibitors of mouse P4502b and rat P4502B1. 2-EN was a 5- to 10-fold less potent inhibitor of mouse P4502b, as compared with the rat, even though it was shown to bind to the active site of the mouse isoform as demonstrated by its metabolism to 2-naphthylacetic acid. These data suggest that the active site of the mouse P4502b enzyme is functionally similar to the rat P4502B isoform, with the exception of the disparity in its susceptibility to inactivation by 2-EN as measured by the Ki values.

Anti-tumor efficacy of naked siRNAs for ERBB3 or AKT2 against lung adenocarcinoma cell xenografts.

The use of siRNAs against specific molecular targets has potential for cancer therapy but has been thought to be limited by the need for formulation to improve cellular uptake. Lung adenocarcinoma cells are markedly suppressed in culture by siRNAs to the receptor ERBB3 or its downstream signaling partner AKT2. We now demonstrate that naked, unformulated siRNAs to ERBB3 or AKT2, administered i.v. as saline solutions, 2 μg/g five times per week for 3 weeks (total dose 30 μg/g), were effective suppressors of growth of A549 human lung adenocarcinoma cell xenografts in athymic mice, 12 mice per group, in four different experiments. ERBB3 and AKT2 siRNAs each inhibited growth by 70–90% on average, compared to saline‐treated or untreated controls; a nonsilencing siRNA was without significant effect. Lesser but significant effects were noted with a total dose of 12 μg/g. With the higher dose, effects persisted for several weeks after the end of treatment. Expected reductions of ERBB3 and AKT2 mRNAs and proteins occurred and correlated with decrease in tumor volume. There were no significant changes in serum cytokines. These results show that naked siRNAs to ERBB3 or AKT2 may have potential for lung cancer therapy.

Molecular and organismal changes in offspring of male mice treated with chemical stressors

Both gene methylation changes and genetic instability have been noted in offspring of male rodents exposed to radiation or chemicals, but few specific gene targets have been established. Previously, we identified the gene for ribosomal RNA, rDNA, as showing methylation change in sperm of mice treated with the preconceptional carcinogen, chromium(III) chloride. rDNA is a critical cell growth regulator. Here, we investigated the effects of paternal treatments on rDNA in offspring tissue. A total of 93 litters and 758 offspring were obtained, permitting rigorous mixed‐effects models statistical analysis of the results. We show that the offspring of male mice treated with Cr(III) presented increased methylation in a promoter sequence of the rDNA gene, specifically in lung. Furthermore polymorphic variants of the multi‐copy rDNA genes displayed altered frequencies indicative of structural changes, as a function of both tissue type and paternal treatments. Organismal effects also occurred: some groups of offspring of male mice treated with either Cr(III) or its vehicle, acidic saline, compared with those of untreated mice, had altered average body and liver weights and levels of serum glucose and leptin. Males treated directly with Cr(III) or acidic saline presented serum hormone changes consistent with a stress response. These results establish for the first time epigenetic and genetic instability effects in a gene of central physiological importance, in offspring of male mice exposed preconceptionally to chemicals, possibly related to a stress response in these males.

Abstract 184: Stress-induced father-mediated 45S rRNA genetic and epigenetic reprogramming

Environmental and dietary factors modify genomes and phenotypes. The extents of these modifications are not clear. DNA methylation and genotypes of the multi-copy 45S rRNA gene were quantified in 4 tissues at 3 developmental stages (sperm of 128 treated males, 98 litters of 876 day-8 embryos, and lung, liver, and a subset of sperm from 93 litters of 758 6-week adult offspring) in 3 treatment groups. Mixed models for adjusting confounding factors were used for all statistical analyses. Single injections of 1 mmol/kg chromium(III), [Cr(III)], an environmental agent and a dietary supplement, or acid saline (AS) vehicle were given to male mice only, which were then euthanized or bred 2 weeks later. A trend toward hypomethylation in the rRNA spacer promoter region but no frequency differences in 5 sequence variants of the rRNA, defined by 4 single nucleotide polymorphisms, were observed in sperm 2 weeks after Cr(III) treatment as compared to AS and untreated (UT) groups. This epigenetic trend disappeared at the day-8 embryo stage for both genders after paternal treatments with Cr(III) and/or AS. In offspring at 6 weeks of age, significant hypermethylation of the rRNA spacer-promoter was detected in the lung of the male offspring from Cr(III)-treated fathers, reversing the hypomethylation trend seen in the sperm 2 weeks after Cr(III) treatment. There was also a change in genotype: a significant reduction of the rRNA CGC variant in Cr(III) and/or AS groups in lung and liver in males, and in lung in females. Further examinations of the regressions of individual sequence variants on DNA methylation and on other variants revealed significant modifications to those correlates by Cr(III) and/or AS but to different degrees depending on the developmental stages, thus supporting the hypothesis of paternal exposure-induced epigenetic and genetic reprogramming. These genomic reprogrammings were accompanied by phenotypic changes related to paternal Cr(III) or AS treatment, including increased body and liver weights and alterations in serum glucose and leptin. Male mice treated with either Cr(III) or AS demonstrated a typical chemical stress response, as indicated by acute reduction in serum insulin and leptin, followed later by increases in these hormones. Taken together, this multi-faceted cross-generational study uncovers modifiable epigenetic and genetic reprogramming during development and differentiation. Paternal stress apparently had multiple effects on genomes and phenotypes, in their offspring. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 184.