Spontaneous Tumor Development Research Articles

Effect of the mitochondria-targeted antioxidant SkQ1 on development of spontaneous tumors in BALB/c mice.

The mitochondria-targeted antioxidant SkQ1 (10-(6'-plastoquinonyldecyl)triphenylphosphonium) is a new pharmaceutical substance with a wide spectrum of effects including increase in lifespan of laboratory animals (for example, of BALB/c mice males) and inhibition of development of some experimental tumors and also of tumor cell growth. In this work, the effects of SkQ1 on development of spontaneous tumors in female and male BALB/c mice housed in an SPF-class vivarium were studied. We found that the addition of SkQ1 to drinking water at the dose of 1 and 30 nmol/kg body weight per day throughout the lifespan modified the spectrum of spontaneous tumors in the female mice, decreasing the incidence of follicular lymphomas. SkQ1 at the dose of 1 nmol/kg per day also suppressed the dissemination of these neoplasms, but it did not significantly influence the overall incidence of benign and malignant tumors (including primary multiple tumors) or the lifespan of the tumor-bearing mice (both males and females). Hence, the previously described ability of SkQ1 to increase the lifespan of laboratory BALB/c mice is not related to its anticarcinogenic activity.

Abstract 72: Analyzing the role of Astrocyte Elevated Gene-1 (AEG-1) in hepatocarcinogenesis using a knockout mouse model

Abstract The oncogene Astrocyte Elevated Gene-1 (AEG-1) is overexpressed in a wide variety of human cancers. In vitro and in vivo nude mouse xenograft studies have established the tumor-promoting role of AEG-1. Activation of multiple signaling pathways, such as NF-κB, PI3K/Akt, Wnt/β-catenin and MEK/ERK, has been attributed to mediate oncogenesis by AEG-1. To better understand the role AEG-1 plays in the onset and progression of cancer and to identify key mechanism(s) of AEG-1 action, we generated a novel AEG-1 knockout mouse (AEG-1-/-). We studied hepatocarcinogenesis in this model because AEG-1 is overexpressed in more than 90% of human hepatocellular carcinoma (HCC) patients and is believed to play a key regulatory role in progression of the disease. AEG-1-/- mice were viable with normal development. We first compared the aging associated phenotype of WT and AEG-1-/- mice. At 16 months of age spontaneous tumor development in lungs and liver was observed in some cohorts of untreated WT mice (n=20). Such tumorigenesis was not detected in any of the AEG-1-/- mice. Additionally, age-associated inflammatory infiltrates were observed in the internal organs of WT but not AEG-1-/- mice. To initiate HCC, WT and AEG-1-/- mice were treated with diethylnitrosamine (DEN) at 2 weeks of age. At 32 weeks WT mice showed a marked hepatocarcinogenic response while AEG-1-/- mice exhibited profound resistance to tumor formation. The hepatocarcinogenic response was further evaluated by DEN initiation followed by daily phenobarbital treatment. WT mice exhibited an intensified hepatocarcinogenic response evidenced by large necrotic liver tumors at 28 weeks of age with a 52% rate of lung metastasis. AEG-1-/- mice remained remarkably resistant even to this combinatorial treatment with no distant metastasis. No difference in the activation of Akt, ERK and β-catenin signaling was observed between WT and AEG-1-/- mice indicating that these pathways may not be regulated by AEG-1 under physiological conditions. However, marked inhibition of NF-κB activation was observed in AEG-1-/- mice. Lipopolysaccharide (LPS)-induced NF-κB luciferase reporter activity, nuclear translocation of p65 subunit of NF-κB and induction of IL-1β and IL-6 were markedly attenuated in primary hepatocytes and macrophages isolated from AEG-1-/- versus WT mice. Chronic inflammation and NF-κB activation play a causal role in hepatocarcinogenesis. Inhibition of NF-κB activation in hepatocytes and macrophages profoundly abrogates HCC development in mouse models. Our findings in AEG-1-/- mice confirm a fundamental role of AEG-1 in NF-κB activation. Here we demonstrate that lack of AEG-1 protects from generalized inflammation thereby suppressing spontaneous as well as experimental tumorigenesis. AEG-1-/-mice might be a useful model to study inflammation and cancer in diverse organ systems. Citation Format: Chadia L. Robertson, Jyoti Srivastava, Ayesha Siddiq, Rachel Gredler, Devaraja Rajasekaran, Maaged Akiel, Xue-Ning Shen, Knarik Arkun, Shobha Ghosh, Mark A. Subler, Jolene Windle, Paul B. Fisher, Devanand Sarkar. Analyzing the role of Astrocyte Elevated Gene-1 (AEG-1) in hepatocarcinogenesis using a knockout mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 72. doi:10.1158/1538-7445.AM2014-72

Abstract SY40-04: Immune control of spontaneous tumor development

Abstract Long recognized as an evolutionarily ancient cell type to control development, tissue homeostasis, and immune responses to pathogens, macrophages are being rediscovered as regulators of several chronic disorders including cancer. Using a spontaneous mammary tumor model, here we show that tumor growth induces the differentiation and expansion of a unique subset of tumor-associated macrophages (TAMs) that are phenotypically and functionally distinct from mammary tissue macrophages (MTMs). Compared to MTMs, TAMs do not exhibit an “alternatively activated macrophage” phenotype, but express the adhesion molecule Vcam1, and have high proliferative capacity upon their differentiation from CCR2-expressing inflammatory monocytes. Importantly, TAM differentiation is dependent on the transcriptional regulator of the Notch signaling pathway, RBPJ; and TAM, but not MTM, depletion restores tumor-infiltrating cytotoxic T cell responses, and suppresses tumor growth. These findings demonstrate that TAMs represent a novel lineage of mononuclear phagocytes with critical regulatory functions in promoting T cell tolerance to tumors. Citation Format: Ming Li. Immune control of spontaneous tumor development. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY40-04. doi:10.1158/1538-7445.AM2014-SY40-04

71. A novel combined psychosocial stress paradigm alters tumor VEGF and circulating exosomes in a spontaneous, metastatic model of breast cancer

Metastatic breast cancer carries significant risk of morbidity and mortality with limited treatment options. Psychosocial stress exposure has been linked to tumor growth and metastasis; therefore understanding the underlying neurohormonal mechanisms may yield new therapeutic options and lead to the development of more effective disease interventions. Here we investigated the impact of social isolation combined with acute restraint stress on spontaneous tumor development in MMTV-PyMT mice. Mammary tumor progression in this mouse model of metastatic breast cancer mimics that of invasive ductal carcinoma in humans. Female MMTV-PyMT mice were singly housed at 8 weeks of age during the hyperplastic stage of tumor development. Restraint stress was initiated after 2 weeks of isolation. Control mice remained in their home cages throughout the experiment. Twenty-four hours after the final restraint exposure, stressed mice exhibited elevated circulating corticosterone and splenic normetanephrine (a norepinephrine metabolite), which correlated with increased tumor VEGF and decreased splenic neutrophils. There was no difference in total tumor burden at this early carcinoma stage. Circulating CD63+ exosomes, extracellular vesicles that drive cancer pathogenesis, were decreased. These results demonstrate the dual stressor elicited potent hypothalamic pituitary axis (HPA) and sympathetic nervous system (SNS) activation in conjunction with alterations in the premetastatic tumor that may facilitate progression.

Vaccination with ErbB-2 peptides prevents cancer stem cell expansion and suppresses the development of spontaneous tumors in MMTV-PyMT transgenic mice.

ErbB-2 has been implicated as a target for cancer-initiating cells in breast and other cancers. ErbB-2-directed peptide vaccines have been shown to be effective in prevention of spontaneous tumorigenesis of breast in neu transgenic mouse model, and cellular immunity is proposed as a mechanism for the anti-tumor efficacy. However, there has been no explanation as to how immunity suppresses tumorigenesis from the early stage carcinogenesis, when ErbB-2 expression in breast is low. Here, we investigated a peptide-based vaccine, which consists of two MHC class II epitopes derived from murine ErbB-2, to prevent the occurrence of spontaneous tumors in breast and assess immune impact on breast cancer stem cells. Female MMTV-PyMT transgenic mice were immunized with either ErbB-2 peptide vaccine, or a peptide from tetanus toxoid, or PBS in immune adjuvant. ErbB-2 peptides vaccine completely suppressed spontaneous breast tumors, and the efficacy was correlated with antigen-specific T-cell and antibody responses. In addition, immune serum from the mice of ErbB-2 vaccine group had an inhibitory effect on mammosphere-forming capacity and signaling through ErbB-2 and downstream Akt pathway in ErbB-2 overexpressing mouse mammary cancer cells. We provide evidence that multi-epitope class II peptides vaccine suppresses tumorigenesis of breast potentially by inhibiting the growth of cancer stem cells. We also suggest that a strategy of inducing strong immune responses using multi-epitope ErbB-2-directed helper vaccine might be useful in preventing breast cancer recurrence.

P19Ink4d Is a Tumor Suppressor and Controls Pituitary Anterior Lobe Cell Proliferation

Pituitary tumors develop in about one-quarter of the population, and most arise from the anterior lobe (AL). The pituitary gland is particularly sensitive to genetic alteration of genes involved in the cyclin-dependent kinase (CDK) inhibitor (CKI)-CDK-retinoblastoma protein (Rb) pathway. Mice heterozygous for the Rb mutation develop pituitary tumors, with about 20% arising from the AL. Perplexingly, none of the CKI-deficient mice reported thus far develop pituitary AL tumors. In this study, we show that deletion of p19(Ink4d) (p19), a CKI gene, in mice results in spontaneous development of tumors in multiple organs and tissues. Specifically, more than one-half of the mutant mice developed pituitary hyperplasia or tumors predominantly in the AL. Tumor development is associated with increased cell proliferation and enhanced activity of Cdk4 and Cdk6 and phosphorylation of Rb protein. Though Cdk4 is indispensable for postnatal pituitary cell proliferation, it is not required for the hyperproliferative pituitary phenotype caused by p19 loss. Loss of p19 phosphorylates Rb in Cdk4(-/-) pituitary AL cells and mouse embryonic fibroblasts (MEFs) and rescues their proliferation defects, at least partially, through the activation of Cdk6. These results provide the first genetic evidence that p19 is a tumor suppressor and the major CKI gene that controls pituitary AL cell proliferation.

Systems toxicology approaches enable mechanistic comparison of spontaneous and cigarette smoke-related lung tumor development in the A/J mouse model.

The A/J mouse is highly susceptible to lung tumor induction and has been widely used as a screening model in carcinogenicity testing and chemoprevention studies. However, the A/J mouse model has several disadvantages. Most notably, it develops lung tumors spontaneously. Moreover, there is a considerable gap in our understanding of the underlying mechanisms of pulmonary chemical carcinogenesis in the A/J mouse. Therefore, we examined the differences between spontaneous and cigarette smoke-related lung tumors in the A/J mouse model using mRNA and microRNA (miRNA) profiling. Male A/J mice were exposed whole-body to mainstream cigarette smoke (MS) for 18 months. Gene expression interaction term analysis of lung tumors and surrounding non-tumorous parenchyma samples from animals that were exposed to either 300 mg/m3 MS or sham-exposed to fresh air indicated significant differential expression of 296 genes. Ingenuity Pathway Analysis® (IPA®) indicated an overall suppression of the humoral immune response, which was accompanied by a disruption of sphingolipid and glycosaminoglycan metabolism and a deregulation of potentially oncogenic miRNA in tumors of MS-exposed A/J mice. Thus, we propose that MS exposure leads to severe perturbations in pathways essential for tumor recognition by the immune system, thereby potentiating the ability of tumor cells to escape from immune surveillance. Further, exposure to MS appeared to affect expression of miRNA, which have previously been implicated in carcinogenesis and are thought to contribute to tumor progression. Finally, we identified a 50-gene expression signature and show its utility in distinguishing between cigarette smoke-related and spontaneous lung tumors.

Mammary gland-specific ablation of focal adhesion kinase reduces the incidence of p53-mediated mammary tumour formation.

Background:Elevated expression of focal adhesion kinase (FAK) occurs in numerous human cancers including colon-, cervix- and breast cancer. Although several studies have implicated FAK in mammary tumour formation induced by ectopic oncogene expression, evidence supporting a role for FAK in spontaneous mammary tumour development caused by loss of tumour suppressor genes such as p53 is lacking. Alterations in the tumour suppressor gene p53 have been implicated in over 50% of human breast cancers. Given that elevated FAK expression highly correlates with p53 mutation status in human breast cancer, we set out to investigate the importance of FAK in p53-mediated spontaneous mammary tumour development.Methods:To directly assess the role of FAK, we generated mice with conditional inactivation of FAK and p53. We generated female p53lox/lox/FAK+/+/WapCre, p53lox/lox/FAKflox/+/WapCre and p53lox/lox/FAKflox/−/WapCre mice, and mice with WapCre-mediated conditional expression of p53R270H, the mouse equivalent of human p53R273H hot spot mutation, together with conditional deletion of FAK, P53R270H/+/FAKlox/+/WapCre and p53R270H/+/FAKflox/−/WapCre mice. All mice were subjected to one pregnancy to induce WapCre-mediated deletion of p53 or expression of p53 R270H, and Fak genes flanked by two loxP sites, and subsequently followed the development of mammary tumours.Results:Using this approach, we show that FAK is important for p53-induced mammary tumour development. In addition, mice with the mammary gland-specific conditional expression of p53 point mutation R270H, the mouse equivalent to human R273H, in combination with conditional deletion of Fak showed reduced incidence of p53R270H-induced mammary tumours. In both models these effects of FAK were related to reduced proliferation in preneoplastic lesions in the mammary gland ductal structures.Conclusions:Mammary gland-specific ablation of FAK hampers p53-regulated spontaneous mammary tumour formation. Focal adhesion kinase deletion reduced proliferative capacity of p53 null and p53R270H mammary epithelial cells but did not lead to increased apoptosis in vivo. Our data identify FAK as an important regulator in mammary epithelial cell proliferation in p53-mediated and p53R270H-induced mammary tumour development.

DNA damage response and DNA repair – dog as a model?

BackgroundCompanion animals like dogs frequently develop tumors with age and similarly to human malignancies, display interpatient tumoral heterogeneity. Tumors are frequently characterized with regard to their mutation spectra, changes in gene expression or protein levels. Among others, these changes affect proteins involved in the DNA damage response (DDR), which served as a basis for the development of numerous clinically relevant cancer therapies. Even though the effects of different DNA damaging agents, as well as DDR kinetics, have been well characterized in mammalian cells in vitro, very little is so far known about the kinetics of DDR in tumor and normal tissues in vivo.DiscussionDue to (i) the similarities between human and canine genomes, (ii) the course of spontaneous tumor development, as well as (iii) common exposure to environmental agents, canine tumors are potentially an excellent model to study DDR in vivo. This is further supported by the fact that dogs show approximately the same rate of tumor development with age as humans. Though similarities between human and dog osteosarcoma, as well as mammary tumors have been well established, only few studies using canine tumor samples addressed the importance of affected DDR pathways in tumor progression, thus leaving many questions unanswered.SummaryStudies in humans showed that misregulated DDR pathways play an important role during tumor development, as well as in treatment response. Since dogs are proposed to be a good tumor model in many aspects of cancer research, we herein critically investigate the current knowledge of canine DDR and discuss (i) its future potential for studies on the in vivo level, as well as (ii) its possible translation to veterinary and human medicine.

Decreased mitochondrial SIRT3 expression is a potential molecular biomarker associated with poor outcome in breast cancer.

SIRT3 is a genomically expressed, mitochondrial localized tumor suppressor protein where it directs multiple metabolic processes by deacetylating downstream protein substrates. Genetic deletion of Sirt3 in mice leads to the spontaneous development of mammary tumors starting at 1 year, and decreased SIRT3 messenger RNA has been observed in several human tumors including breast malignancies. In this investigation, we assessed SIRT3 expression in human breast cancer tissue microarray and examined the relationship between SIRT3 expression and outcome in patients with breast cancer. SIRT3 protein expression is significantly lower in neoplastic compared with normal breast epithelium, including normal epithelium adjacent to tumors. Patients with breast cancer in the lowest quartile of SIRT3 expression had a significantly shorter locoregional relapse-free survival (hazard ratio, 0.53 [0.47-0.61]; log-rank P = 0). Notably, low SIRT3 expression was associated with reduced locoregional relapse-free survival in all breast cancer subtypes analyzed, including ER+, ER-, HER2+, and basal subtypes (hazard ratios, 0.44-0.65; log-rank P = 0-.0019). These results highlight the importance of the SIRT3 as a tumor suppressor protein in breast cancer and suggest that SIRT3 may be a potential molecular biomarker to identify high-risk patients across all molecular subtypes of breast cancer.

Rapid target gene validation in complex cancer mouse models using re‐derived embryonic stem cells

Human cancers modeled in Genetically Engineered Mouse Models (GEMMs) can provide important mechanistic insights into the molecular basis of tumor development and enable testing of new intervention strategies. The inherent complexity of these models, with often multiple modified tumor suppressor genes and oncogenes, has hampered their use as preclinical models for validating cancer genes and drug targets. In our newly developed approach for the fast generation of tumor cohorts we have overcome this obstacle, as exemplified for three GEMMs; two lung cancer models and one mesothelioma model. Three elements are central for this system; (i) The efficient derivation of authentic Embryonic Stem Cells (ESCs) from established GEMMs, (ii) the routine introduction of transgenes of choice in these GEMM-ESCs by Flp recombinase-mediated integration and (iii) the direct use of the chimeric animals in tumor cohorts. By applying stringent quality controls, the GEMM-ESC approach proofs to be a reliable and effective method to speed up cancer gene assessment and target validation. As proof-of-principle, we demonstrate that MycL1 is a key driver gene in Small Cell Lung Cancer.

Bile acid accelerates erbB2-induced pro-tumorigenic activities in biliary tract cancer.

Although very few studies have addressed the molecular and cellular mechanisms underlying the development of biliary tract cancer (BTC), several lines of evidence suggest a role for the erbB receptor family. Overexpression and activation of erbB2 has been reported in a significant percentage of human BTC. Further, we previously reported that overexpression of erbB2 basal epithelial cells of the biliary tract (BK5.erbB2 mouse) led to the development of BTC. However, the mechanisms by which erbB2 overexpression led to the spontaneous development of tumors specifically in the biliary tract are not completely understood. The goals of the current study were to (1) determine whether a cooperative relationship between bile acid exposure and erbB2 activation exists during biliary tract carcinogenesis and (2) to characterize the mechanism(s) underlying bile acid-mediated biliary tract carcinogenesis in cells with activated erbB2. In this study, we demonstrated that the secondary conjugated bile acid, taurochenodeoxycholic acid (TCDC), increased proliferation of primary cultured gallbladder epithelial cells from BK5.erbB2 mice and human BTC cells. TCDC treatment activated EGFR/erbB2 and downstream signaling molecules in both primary cultured cells and human BTC cells. TCDC also increased the expression of epidermal growth factor receptor (EGFR) ligands and TACE activity in human BTC cells. Inhibition of src activation led to attenuation of bile-induced upregulation of TACE activity as well as signaling through the EGFR/erbB2, suggesting that during the development of BTC erbB2 overexpression/activation accelerates the bile acid-induced signaling cascade: bile acid → src → TACE → EGFR/erbB2 → downstream signaling. We also provide direct evidence that bile acids possess tumor promoting capacity in epithelial cells overexpressing erbB2 using the two-stage skin carcinogenesis model. Collectively these findings suggest cooperative roles for bile acid and erbB2 activation in epithelial cell proliferation; bile acid appears to accelerate erbB2-induced pro-tumorigenic activities in the biliary tract and skin.

Impact of Obesity on Development and Progression of Mammary Tumors in Preclinical Models of Breast Cancer

Overweight and/or obesity are known risk factors for postmenopausal breast cancer. More recently increased body weight has also been associated with poor prognosis for premenopausal breast cancer. This relationship has primarily been identified through epidemiological studies. Additional information from in vitro studies has also been produced in attempts to delineate mechanisms of action for the association of obesity and body weight and breast cancer. This approach has identified potential growth factors such as insulin, leptin, estrogen and IGF-I which are reported to be modulated by body weight changes. However, in vitro studies are limited in scope and frequently use non-physiological concentrations of growth factors, while long follow-up is needed for human studies. Preclinical animal models provide an intermediary approach to investigate the impact of body weight and potential growth factors on mammary/breast tumor development and progression. Here results of a number of studies addressing this issue are presented. In the majority of the studies either genetically-obese or diet-induced obese rodent models have been used to investigate spontaneous, transgenic and carcinogen-induced mammary tumor development. To study tumor progression the major focus has been allograft studies in mice with either genetic or dietary-induced obesity. In general, obesity has been demonstrated to shorten mammary tumor latency and to impact tumor pathology. However, in rodents with defects in leptin and other growth factors the impact of obesity is not as straightforward. Future studies using more physiologically relevant obesity models and clearly distinguishing diet composition from body weight effects will be important in continuing to understand the factors associated with body weight's impact on mammary/breast cancer development and progression.

Light pollution increases morbidity and mortality rate from different causes in female rats

The influence of different light regimes (constant light--LL; constant darkness--DD; standard light regime--LD, 12 hours light 12 hours darkness; natural lightening of the North-West of Russia--NL) on the dynamics of life's morbidity rate, spontaneous tumorigenesis and frequency of some kinds of non-tumor pathology revealed at the post-mortem examination of male rats was studied. It was found out that the maintenance of animals at LL and NL conditions led to the increase of the number of infectious diseases, substantially faster development of spontaneous tumors and the increase of non-tumor diseases in comparison with the animals kept at LD (standard light) regime. Light deprivation (DD) led to substantial reduction of development of new growth, of non-tumor and infectious diseases in comparison with the similar parameters in standard light regime.

Exposure to light at night accelerates aging and spontaneous uterine carcinogenesis in female 129/Sv mice

The effect of the constant illumination on the development of spontaneous tumors in female 129/Sv mice was investigated. Forty-six female 129/Sv mice starting from the age of 2 mo were kept under standard light/dark regimen [12 h light (70 lx):12hr dark; LD, control group], and 46 of 129/Sv mice were kept under constant illumination (24 h a day, 2,500 lx, LL) from the age of 5 mo until to natural death. The exposure to the LL regimen significantly accelerated body weight gain, increased body temperature as well as acceleration of age-related disturbances in estrous function, followed by significant acceleration of the development of the spontaneous uterine tumors in female 129/Sv mice. Total tumor incidence as well as a total number of total or malignant tumors was similar in LL and LD group (p > 0.05). The mice from the LL groups survived less than those from the LD group (χ2 = 8.5; p = 0.00351, log-rank test). According to the estimated parameters of the Cox’s regression model, constant light regimen increased the relative risk of death in female mice compared with the control (LD) group (p = 0.0041). The data demonstrate in the first time that the exposure to constant illumination was followed by the acceleration of aging and spontaneous uterine tumorigenesis in female 129/Sv mice.

The protein poly(ADP-ribosyl)ation system: Its role in genome stability and lifespan determination

The processes that lead to violation of genome integrity are known to increase with age. This phenomenon is caused both by increased production of reactive oxygen species and a decline in the efficiency of antioxidant defense system as well as systems maintaining genome stability. Accumulation of different unrepairable genome damage with age may be the cause of many age-related diseases and the development of phenotypic and physiological signs of aging. It is also clear that there is a close connection between the mechanisms of the maintenance of genome stability, on one hand, and the processes of spontaneous tumor formation and lifespan, on the other. In this regard, the system of protein poly(ADP-ribosyl)ation activated in response to a variety of DNA damage seems to be of particular interest. Data accumulated to date suggest it to be a kind of focal point of cellular processes, guiding the path of cell survival or death depending on the degree of DNA damage. This review summarizes and analyzes data on the involvement of poly(ADP-ribosyl)ation in various mechanisms of DNA repair, its interaction with progeria proteins, and the possible role in the development of spontaneous tumors and lifespan determination. Special attention is given to the relationship between various polymorphisms of the human poly(ADP-ribose) polymerase-1 gene and longevity.

P53 Efficiently Suppresses Tumor Development in the Complete Absence of Its Cell-Cycle Inhibitory and Proapoptotic Effectors p21, Puma, and Noxa

Activation of apoptosis through transcriptional induction of Puma and Noxa has long been considered to constitute the critical (if not sole) process by which p53 suppresses tumor development, although G1/S boundary cell-cycle arrest via induction of the CDK inhibitor p21 has also been thought to contribute. Recent analyses of mice bearing mutations that impair p53-mediated induction of select target genes have indicated that activation of apoptosis and G1/S cell-cycle arrest may, in fact, be dispensable for p53-mediated tumor suppression. However, the expression of Puma, Noxa, and p21 was not abrogated in these mutants, only reduced; therefore, the possibility that the reduced levels of these critical effectors of p53-mediated apoptosis and G1/S-cell-cycle arrest sufficed to prevent tumorigenesis could not be excluded. To resolve this important issue, we have generated mice deficient for p21, Puma, and Noxa (p21-/-puma-/-noxa-/- mice). Cells from these mice were deficient in their ability to undergo p53-mediated apoptosis, G1/S cell-cycle arrest, and senescence. Nonetheless, these animals remained tumor free until at least 500 days, in contrast to p53-deficient mice, which had all succumbed to lymphoma or sarcoma by 250 days. Interestingly, DNA lesions induced by γ-irradiation persisted longer in p53-deficient cells compared to wild-type or p21-/-puma-/-noxa-/- cells, and the former failed to transcriptionally activate several p53 target genes implicated in DNA repair. These results demonstrate beyond a doubt that the induction of apoptosis, cell-cycle arrest, and possibly senescence is dispensable for p53-mediated suppression of spontaneous tumor development and indicate that coordination of genomic stability and possibly other processes, such as metabolic adaptation, may instead be critical.

Effect of cytokinins on expression of radish CLE genes

CLE (CLAVATA3/ENDOSPERM SURROUNDING REGION) peptides are peptide phytohormones playing the important role in the regulation of various type meristem development and also in the interaction between plants and parasites and symbionts. At the same time, the interaction of CLE peptides with other phytohormones, participating in these processes, is essentially unstudied. Using real-time RT-PCR, we analyzed expression of some genes encoding CLE peptides in radish (Raphanus sativus var. radicula Pers.) under normal conditions and after treatment with the cytokinin 6-benzyladenine. Even after short-term (30 min) action of cytokinin, expression of group A CLE genes was manifold suppressed, whereas cytokinin did not affect expression of group B CLE genes. Radish lines contrasting in the trait “spontaneous tumor formation” demonstrated similar dynamics of CLE gene expression in response to cytokinin treatment but differed in the levels of their expression. A possibility of interaction between CLE peptides and cytokinins in the spontaneous development of tumors in radish lines is discussed. The important part of the work was the selection of optimal reference genes for the analysis of radish gene expression by real-time RT-PCR method. Most stable expression was observed for the genes of ubiquitin (RsUBQ) and glyceraldehydes-3-phosphate dehydrogenase (RsGAPDH).

Abstract 3852: Bcl-xL and IL-6 act independently and synergistically to accelerate plasmacytopoiesis and plasma cell tumor formation in Balb/c mice.

Abstract Accelerated development of spontaneous plasma cell tumors (PCT) occurs in mucosal-associated lymphoid tissues of mice bearing both IL-6 and BCL-XL transgenes (double transgenic (dTg)). 91% of dTG mice developed PCTs at a mean age of 89 days. In contrast, 53% of the IL-6 Tg mice developed PCTs at a mean age of 301 days and 12% BCL-XL Tg mice developed PCTs with a mean age of 433 days. The PCTs formed primarily in Peyer's Patches, medullary cords of the mesenteric lymph node and/or the lamina propria of small intestinal villi where they were preceded by massive plasma cell hyperplasia. To investigate the molecular basis of the cooperation between IL-6 and BCL-xL transgenes to accelerate PCT development we performed detailed analysis of cell lines developed from dTG mice and and spontaneous or pristane-induced tumors of Bcl-xL TG mice. All the tumors carry Ig/Myc-deregulating chromosomal translocations, which are thought to be the initiating event of PCT development. Therefore we focused on finding possible differences in secondary alterations. Although the subsets were poorly distinguished by microarray-based gene expression profiling, there were significant differences in the expression of p21, p19ARF, p53, HUWE1 and pRb as determined by western blotting and qPCR. DTG tumors were characterized by alterations in pathways governing expression of pRb that included increased expression of INK4A and down regulation of pRb itself as well as down regulation of p19ARF. Most notably we discovered discordance between protein and mRNA levels for pRb and p19Arf. Efforts to understand the molecular basis for these distinctions and their contributions to the biology of these PCT subsets are in progress. Citation Format: Tomomi Sakai, Alexander L. Kovalchuk, Herbert Morse. Bcl-xL and IL-6 act independently and synergistically to accelerate plasmacytopoiesis and plasma cell tumor formation in Balb/c mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3852. doi:10.1158/1538-7445.AM2013-3852

Abstract 174: Calorie restriction decreases spontaneous mammary tumorigenesis and upregulates ER-β in MMTV-neu transgenic mice.

Abstract Obesity increases postmenopausal breast cancer risk and increases mortality in pre- and postmenopausal women. While the majority of breast cancers in obese women are estrogen receptor (ERα)-positive, ERα-negative tumors confer a much worse prognosis. The mechanism by which obesity affects ER-negative breast cancer risk and prognosis is not clear, and strategies for offsetting the negative effects of obesity are urgently needed. In this study, we utilized the MMTV-neu mouse model of luminal-type breast cancer to test the hypothesis that energy balance modulation, through diet-induced obesity (DIO) or calorie restriction (CR) regimens, alters mammary tumor development and progression through regulation of ER in the mammary epithelium. MMTV-neu mice form spontaneous mammary tumors that progress from an ERα-positive hyperplasia to aggressive ERα-negative ductal adenocarcinomas. Female MMTV-neu transgenic mice and non-transgenic host strain (FVB) mice (6-8 weeks old; n=90/genotype) were randomized (30/group) to receive: control diet (modified AIN-76A); a 30% CR regimen (isonutrient); or a DIO regimen. A subset of mice (n=4 per group) was killed at 1, 3, and 5 months following diet initiation, and tissues were collected for analysis; remaining animals were followed for a 60-week survival study. We found that, relative to control diet, the DIO regimen significantly increased body weight, percent body fat (p<0.0001), and obesity-associated serum hormones and growth factors (IGF-I, insulin, leptin, and estradiol; p<0.01 for all) in both MMTV-neu and FVB controls. Conversely, CR significantly decreased body weight, percent body fat (p<0.0001) and decreased serum hormones/ growth factors, while increasing adiponectin (each at p<0.01). Gene expression (qRT-PCR) and protein expression (immunohistochemistry) analysis revealed that loss of mammary ERα expression, known to occur by 8 weeks of age in control MMTV-neu mice, was accelerated by DIO and delayed by CR. Additionally, we found that CR (relative to control diet) significantly increased mammary ERβ expression (p<0.0001) and delayed the onset of hyperplasia (p<0.001) in both MMTV-neu and FVB mice. Importantly, we found that after 60 weeks of feeding, CR significantly increased tumor-free survival of MMTV-neu mice (p=0.01). CR tumor samples showed a decrease in vascularity and presence of mitotic figures. In conclusion, dietary energy balance modulation impacts spontaneous MMTV-neu mammary tumor development and ERα and ERβ levels in normal and tumor tissue. In addition, increased mammary ERβ expression may represent a novel mechanism underlying the anticancer effects of CR. Citation Format: Sarah M. Dunlap, Nikki A. Ford, Laura W. Bowers, Emily L. Rossi, Samantha Miller, Lucia J. Chiao, Powel H. Brown, Stephen D. Hursting. Calorie restriction decreases spontaneous mammary tumorigenesis and upregulates ER-β in MMTV-neu transgenic mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 174. doi:10.1158/1538-7445.AM2013-174