Tumor Development In Mice Research Articles

Abstract LB-8: A novel function of Vps34 in regulation of cell proliferation

Abstract Many new insights into the biology of class III PI3Kinase have recently been reported. Most of these studies, however, used mainly transformed cells. It is unclear whether class III PI3Kinase works similarly in non-transformed mammalian cells. We explored this by depleting two non-transformed human epithelial cell lines of Vps34, the catalytic subunit of the lipid kinase using shRNA. Interestingly, in a stark contrast to transformed cells where depletion of Vps34 was associated with decreased growth, Vps34 deficiency in non-transformed cells resulted in a marked increase in cell proliferation. The deregulated autophagy function and E-cadherin-dependent cell-cell contacts found in Vps34-depleted cells appeared to be not involved in cell growth regulation as neither silence of Atg7 expression nor inactivation of E-cadherin had any effect on cell proliferation. Reporter luciferase-based ten-pathway screens suggested an altered activity of the MAPK pathway. Indeed, biochemical analysis indicated a sustained ERK activation resulting from the impediment of endosome-mediated EGFR turnover in Vps34 deficient cells. A contribution of the endosomal pathway to Vps34-mediated regulation of cell proliferation was further confirmed by using Rab5 and Rab11 mutants that perturbed early and recycling endosomes, respectively. Importantly, inactivation of Vps34 compromised the cellular barrier against oncogene-mediated transformation, as demonstrated by the increased susceptibility of Vps34 depleted cells to Ras-induced colony formation in vitro and tumor development in mice. Our data collectively uncover a novel role for the class III PI3Kinase-endosomal pathway in regulation of cell proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-8. doi:10.1158/1538-7445.AM2011-LB-8

Effect of Ditaxin and Heteranthin and Inhibitory Effect of Ditaxis heterantha Extract on L5178Y Tumor Development in Mice

Ditaxis heterantha seeds are used as spices for flavoring and coloring food. Two new apocarotenoids derived from the seeds, heteranthin and ditaxin, were evaluated for their in vitro cytotoxic effects in murine lymphoma cells lines. Bioabsorption in mice and preventive and antitumor effects of the apocarotenoids were determined. Ditaxin and heteranthin showed cytotoxic effects in vitro against murine malignant cells and normal splenocyte cells. The 50% inhibitory concentration (IC(50)) for ditaxin in splenocytes was 0.1825 mM; in L5178Y, the IC(50) was 0.1923 mM. The heteranthin IC(50) in splenocytes was 0.1325 mM; in L5178Y, the value was 0.3889 mM. The maximum ditaxin plasma concentration was found after 2 hours of administration (mean±standard deviation, 7.5±2.05 μg/mL). Oral administration of the D. heterantha extract (100 mg/kg per day) for 14 days after the L5178Y lymphoma cell implantation showed no significant effect compared with groups that were not pretreated. However, tumor inhibition in groups treated intraperitoneally before inoculation with the L5178Y cells showed a significant difference (P<.001) compared with the groups not pretreated.

The in-vitro and in-vivo inhibitory activity of biflorin in melanoma

Biflorin, an ortho-naphthoquinone, is an active compound found in the roots of Capraria biflora L. It has been reported that biflorin presents anticancer activity, inhibiting both tumor cell line growth in culture and tumor development in mice. The aim of this study was to examine the effectiveness of biflorin treatment using both in-vitro and in-vivo melanoma models. Biflorin displayed considerable cytotoxicity against all tested cell lines, with half maximal inhibitory concentration values ranging from 0.58 μg/ml in NCI H23 (human lung adenocarcinoma) to 14.61 μg/ml in MDA-MB-231 (human breast cancer) cell lines. In a second set of experiments using B16 melanoma cells as a model, biflorin reduced cell viability but did not cause significant increase in the number of nonviable cells. In addition, the DNA synthesis was significantly inhibited. Flow cytometry analysis showed that biflorin may lead to an apoptotic death in melanoma cells, inducing DNA fragmentation and mitochondria depolarization, without affecting membrane integrity. In B16 melanoma-bearing mice, administration of biflorin (25mg/day) for 10 days inhibited tumor growth, and also increased the mean survival rate from 33.3±0.9 days (control) to 44.5±3.4 days (treated). Our findings suggest that biflorin may be considered as a promising lead compound for designing new drugs to be used in the treatment of melanoma.

Deletion of p120-Catenin Results in a Tumor Microenvironment with Inflammation and Cancer that Establishes It as a Tumor Suppressor Gene

p120-catenin (p120ctn) interacts with E-cadherin, but to our knowledge, no formal proof that p120ctn functions as a bona fide tumor suppressor gene has emerged to date. We report herein that p120ctn loss leads to tumor development in mice. We have generated a conditional knockout model of p120ctn whereby mice develop preneoplastic and neoplastic lesions in the oral cavity, esophagus, and squamous forestomach. Tumor-derived cells secrete granulocyte macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor-α (TNFα). The tumors contain significant desmoplasia and immune cell infiltration. Immature myeloid cells comprise a significant percentage of the immune cells present and likely participate in fostering a favorable tumor microenvironment, including the activation of fibroblasts.

Arsenic trioxide inhibits human cancer cell growth and tumor development in mice by blocking Hedgehog/GLI pathway

The Hedgehog (Hh) pathway is activated in some human cancers, including medulloblastoma. The glioma-associated oncogene homolog (GLI) transcription factors are critical mediators of the activated Hh pathway, and their expression may be elevated in some tumors independent of upstream Hh signaling. Thus, therapies targeting GLI transcription factors may benefit a wide spectrum of patients with mutations at different nodal points of the Hh pathway. In this study, we present evidence that arsenic trioxide (ATO) suppresses human cancer cell growth and tumor development in mice by inhibiting GLI1. Mechanistically, ATO directly bound to GLI1 protein, inhibited its transcriptional activity, and decreased expression of endogenous GLI target genes. Consistent with this, ATO inhibited the growth of human cancer cell lines that depended on upregulated GLI expression in vitro and in vivo in a xenograft model of Ewing sarcoma. Furthermore, ATO improved survival of a clinically relevant spontaneous mouse model of medulloblastoma with activated Hh pathway signaling. Our results establish ATO as a Hh pathway inhibitor acting at the level of GLI1 both in vitro and in vivo. These results warrant the clinical investigation of ATO for tumors with activated Hh/GLI signaling, in particular patients who develop resistance to current therapies targeting the Hh pathway upstream of GLI.

Abstract P5-03-07: Adiponectin Deficiency Accelerated Breast Cancer Development and Promoted Cholesterol Accumulation in Mammary Tumors of MMTV-Polyomavirus Middle T Antigen Mice

Abstract Breast cancer is the most common cancer in women. Visceral obesity and body weight gain are associated with the increased risk of postmenopausal breast cancer. Adiponectin is an adipokine possessing anti-tumor activities against various types of obesity-related cancers. The present study has established a MMTV-polyomavirus middle T antigen (PyVT) transgenic mouse deficient with adiponectin (ADN) expression. Compared to the control mice [PyVT(+/−)ADN(+/+)], the development of mammary tumors in mice lacking this adipokine [PyVT(+/−)ADN(-/-)] was significantly accelerated. While the serum total cholesterol was progressively decreased from week 11 onwards, the cholesterol content in tumor tissues was elevated in both types of mice. The ratio between serum LDL-cholesterol and HDL-cholesterol was significantly higher in PyVT(+/−)ADN(-/-) mice than that in PyVT(+/−)ADN(+/+) mice. Adiponectin was able to inhibit cholesterol uptake in both MDA-MB-231 human breast cancer cells and the primary tumor cells isolated from PyVT transgenic mice. These data demonstrated that the anti-breast cancer activity of adiponectin was at least in part attributed to its suppressive effects on cholesterol accumulation in mammary tumors. This work is supported by Research Grants Council of Hong Kong (Project no.777908M). Adiponectin knockout mice were kindly provided by Dr. Lawrence Chan at Baylor College of Medicine, who generated these mice with the support of the US National Institutes of Health grant HL-51586. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-03-07.

Effects of low level microwave radiation on carcinogenesis in Swiss Albino mice

This study concerns with the multiple treatment of the target site to potent carcinogen and the super imposition of low level radiofrequency and microwave radiation. Swiss albino mice (male) were used for this investigation. The study has been divided in two parts, part A: a single dose of 7,12-dimethylbenz(a)anthracene (DMBA) 100 μg/animal was applied topically on the skin of mice and were exposed to 112 MHz amplitude modulated (AM) at 16 Hz (power density 1.0 mW/cm(2), specific absorption rate (SAR) 0.75 W/kg). Similarly after a single dose of DMBA, mice were exposed to 2.45 GHz radiation (power density of 0.34 mW/cm(2), SAR, 0.1 W/kg), 2 h/day, 3 days a week for a period of 16 weeks. The two sets of experiments were carried out separately. Part B: mice were transplanted intraperitoneally (ip) with ascites 8 × 10(8) (Ehrlich-Lettre ascites, strain E) carcinoma cells per mouse. These mice were exposed to 112 MHz amplitude modulated at 16 Hz and 2.45 GHz radiation separately for a period of 14 days. There was no tumor development in mice exposed to RF and MW. Similarly a topical application of single dose of DMBA followed by RF/MW exposure also did not produce any visible extra tumor on the skin of mice. On the other hand mice were transplanted intraperitoneally with ascites (8 × 10(8) cell/ml) and subsequently exposed to above mentioned fields for 14 days showed a slight increase in the cell numbers as compared to the control group. However, the increase is insignificant. There were insignificant differences either in the mortality or cell proliferation among the control and exposed group. This results show that low level RF or MW do not alter tumor growth and development as evidenced by no observable change in tumor size.

Evidence That Gsta4 Modifies Susceptibility to Skin Tumor Development in Mice and Humans

The incidence of nonmelanoma skin cancer (NMSC) is equivalent to that of all other cancers combined. Previously, we mapped the 12-O-tetradecanoylphorbol-13-acetate (TPA) skin tumor promotion susceptibility locus, Psl1, to distal chromosome 9 in crosses of sensitive DBA/2 mice with relatively resistant C57BL/6 mice. Here, we used the mouse two-stage skin carcinogenesis model to identify the gene(s) responsible for the effects of Psl1. Interval-specific congenic mouse strains (n ≥ 59 mice per strain) were used to more precisely map the Psl1 locus. Having identified glutathione S-transferase α4 (Gsta4) as a candidate tumor promotion susceptibility gene that mapped within the delimited region, we analyzed Gsta4-deficient mice (n = 62) for susceptibility to skin tumor promotion by TPA. We used quantitative polymerase chain reaction, western blotting, and immunohistochemistry to verify induction of Gsta4 in mouse epidermis following TPA treatment and biochemical assays to associate Gsta4 activity with tumor promotion susceptibility. In addition, single-nucleotide polymorphisms (SNPs) in GSTA4 were analyzed in a case-control study of 414 NMSC patients and 450 control subjects to examine their association with human NMSC. Statistical analyses of tumor studies in mice were one-sided, whereas all other statistical analyses were two-sided. Analyses of congenic mice indicated that at least two loci, Psl1.1 and Psl1.2, map to distal chromosome 9 and confer susceptibility to skin tumor promotion by TPA. Gsta4 maps to Psl1.2 and was highly induced (mRNA and protein) in the epidermis of resistant C57BL/6 mice compared with that of sensitive DBA/2 mice following treatment with TPA. Gsta4 activity levels were also higher in the epidermis of C57BL/6 mice following treatment with TPA. Gsta4-deficient mice (C57BL/6.Gsta4(-/-) mice) were more sensitive to TPA skin tumor promotion (0.8 tumors per mouse vs 0.4 tumors per mouse in wild-type controls; difference = 0.4 tumors per mouse; 95% confidence interval = 0.1 to 0.7, P = .007). Furthermore, inheritance of polymorphisms in GSTA4 was associated with risk of human NMSC. Three SNPs were found to be independent predictors of NMSC risk. Two of these were associated with increased risk of NMSC (odds ratios [ORs] = 1.60 to 3.42), while the third was associated with decreased risk of NMSC (OR = 0.63). In addition, a fourth SNP was associated with decreased risk of basal cell carcinoma only (OR = 0.44). Gsta4/GSTA4 is a novel susceptibility gene for NMSC that affects risk in both mice and humans.

Immunomodulatory and antitumor activity of Aerva lanata ethanolic extract

Cancer is responsible for millions of deaths each year worldwide. Pharmacological intervention with plant-derived products alone or in combination to reverse, suppress, or prevent the cancer progression plays a key role in the fight against this terrible disease. Aerva lanata is an important medicinal plant widely used in traditional systems of medicine like ayurveda and siddha. Ethanolic extract of whole plant of A. lanata exhibited immunomodulatory and antitumor activity. Intraperitoneal administration of five doses of the extract (10 mg/kg body weight) was found to enhance the total WBC count (14,238 cells/mm3), bone marrow cellularity (22.33 × 106 cells/femur), and number of α-esterase-positive cells (1276 cells/4000 cells). Aerva treatment also showed enhanced proliferation of splenocytes, thymocytes, and bone marrow cells both in the presence and absence of specific mitogens in vitro and in vivo. The number of plaque-forming cells (PFC) in spleen (243.33 PFC/106 spleen cells) and circulating antibody titer were also increased (P < 0.001). The extract was 100% cytotoxic to Dalton’s lymphoma ascites (DLA) and Ehrlich ascites carcinoma (EAC) cells at a concentration of 500 µg/mL. It was also found to be cytotoxic toward L929 and HELA cells at higher concentrations, whereas the nontoxic concentrations produced a reduction in the rate of proliferation. Simultaneous administration of five doses of A. lanata extract could produce significant inhibition in DLA-induced solid tumor development in mice and increase the life span of mice-bearing EAC tumors by 53.47%.

Helicobacter pylori Infection Promotes Methylation and Silencing of Trefoil Factor 2, Leading to Gastric Tumor Development in Mice and Humans

Trefoil factors (TFFs) regulate mucosal repair and suppress tumor formation in the stomach. Tff1 deficiency results in gastric cancer, whereas Tff2 deficiency increases gastric inflammation. TFF2 expression is frequently lost in gastric neoplasms, but the nature of the silencing mechanism and associated impact on tumorigenesis have not been determined. We investigated the epigenetic silencing of TFF2 in gastric biopsy specimens from individuals with Helicobacter pylori-positive gastritis, intestinal metaplasia, gastric cancer, and disease-free controls. TFF2 function and methylation were manipulated in gastric cancer cell lines. The effects of Tff2 deficiency on tumor growth were investigated in the gp130(F/F) mouse model of gastric cancer. In human tissue samples, DNA methylation at the TFF2 promoter began at the time of H pylori infection and increased throughout gastric tumor progression. TFF2 methylation levels were inversely correlated with TFF2 messenger RNA levels and could be used to discriminate between disease-free controls, H pylori-infected, and tumor tissues. Genome demethylation restored TFF2 expression in gastric cancer cell lines, so TFF2 silencing requires methylation. In Tff2-deficient gp130(F/F)/Tff2(-/-) mice, proliferation of mucosal cells and release of T helper cell type-1 (Th-1) 1 cytokines increased, whereas expression of gastric tumor suppressor genes and Th-2 cytokines were reduced, compared with gp130(F/F)controls. The fundus of gp130(F/F)/Tff2(-/-) mice displayed glandular atrophy and metaplasia, indicating accelerated preneoplasia. Experimental H pylori infection in wild-type mice reduced antral expression of Tff2 by increased promoter methylation. TFF2 negatively regulates preneoplastic progression and subsequent tumor development in the stomach, a role that is subverted by promoter methylation during H pylori infection.

Abstract B29: Genetic interactions between AKT/mTOR and Ras/MAPK pathways in liver cancer pathogenesis: from animal models to targeted therapy

Abstract Introduction: Hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CCC) are deadly malignant liver tumors lacking of efficient treatment options. Akt/mTOR and Ras/MAPK pathways have been implicated in hepatic carcinogenesis. Our study is to define the genetic interactions between Akt/mTOR and Ras/MAPK pathways in liver carcinogenesis. Methods: Activation of Akt/mTOR and Ras/MAPK signaling was assayed in 62 human HCC and 25 CCC samples. An activated Akt (myr-Akt) was stably transfected into the mouse liver alone or together with activated N-Ras (RasV12) via hydrodynamic gene delivery. Molecular and biochemical features of liver lesions were analyzed. A primary cell line (Akt/Ras) was isolated from a tumor co-injected with Akt and Ras protooncogenes. Akt/Ras cells and human HCC cell lines were treated with different mTOR inhibitors, including NVP-BEZ235, PP242 and Rapamcyin, as well as the MEK inhibitor AZD6244. Results: Using human HCC samples, we demonstrated the coordinated activation of Akt/mTOR and Ras/MAPK pathways in subsets of HCC and CCC characterized by poor prognosis. We found that coexpression of myr-Akt with RasV12 synergizes to promote liver tumor development in mice, leading to HCC and CCC formation within 4 to 6 weeks post injection. At cellular level, tumors from Akt/N-Ras co-injection cells are characterized by high proliferation and low apoptotic index, and disrupted expression of genes involved in cell cycle regulation. At the molecular level, liver tumors show high levels of activated Akt/mTOR and Erk cascades. A primary cell line (Akt/Ras) was isolated from a tumor and used to test the response to different mTOR and MEK inhibitors. We found that dual PI3K/mTOR inhibitor NVP-BEZ235 has superior activity inhibiting Akt/Ras cell proliferation with IC50 around 2nM. It also efficiently inhibits phospho-S6, phospho-4EBP1 and cyclin D1 expression in Akt/Ras cells. Furthermore, NVP-BEZ235 synergizes with the MEK inhibitor AZD6244 to dramatically reduce the growth of the Akt/Ras cell line. Equivalent results were obtained in human HCC cell lines with the same inhibitors. Conclusion: Coordinated activation of Akt/mTOR and Ras/MAPK signaling is the hallmark of aggressive subtypes of human HCC and CCC. Activated Akt cooperates with activated Ras/MAPK to rapidly promote liver tumor formation in mice. The Akt/Ras mouse model is a useful pre-clinical system to test anti-neoplastic therapeutic approaches against HCC and CCC in vivo. In addition, our preliminary studies suggest that the combination of a dual PI3K/mTOR inhibitor with a MEK inhibitor may represent a promising novel targeted therapy for treating human HCC harboring activated Akt/mTOR and Ras/MAPK pathways. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B29.

Conditional ablation of Ikkb inhibits melanoma tumor development in mice

Several lines of evidence suggest that tumor cells show elevated activity of the NF-kappaB transcription factor, a phenomenon often resulting from constitutive activity of IkappaB kinase beta (IKKbeta). However, others have found that loss of NF-kappaB activity or IKKbeta is tumor promoting. The role of NF-kappaB in tumor progression is therefore controversial and varies with tumor type. We sought to more extensively investigate the role IKKbeta in melanoma tumor development by specifically disrupting Ikkb in melanocytes in an established mouse model of spontaneous melanoma, whereby HRasV12 is expressed in a melanocyte-specific, doxycycline-inducible manner in mice null for the gene encoding the tumor suppressor inhibitor cyclin-dependent kinase 4/alternative reading frame (Ink4a/Arf). Our results show that Ink4a/Arf-/- mice with melanocyte-specific deletion of Ikkb were protected from HRasV12-initiated melanoma only when p53 was expressed. This protection was accompanied by cell cycle arrest, with reduced cyclin-dependent kinase 2 (Cdk2), Cdk4, Aurora kinase A, and Aurora kinase B expression. Increased p53-mediated apoptosis was also observed, with decreased expression of the antiapoptotic proteins Bcl2 and survivin. Enhanced stabilization of p53 involved increased phosphorylation at Ser15 and reduced phosphorylation of double minute 2 (Mdm2) at Ser166. Together, our findings provide genetic and mechanistic evidence that mutant HRas initiation of tumorigenesis requires Ikkbeta-mediated NF-kappaB activity.

Disrupting Circadian Homeostasis of Sympathetic Signaling Promotes Tumor Development in Mice

BackgroundCell proliferation in all rapidly renewing mammalian tissues follows a circadian rhythm that is often disrupted in advanced-stage tumors. Epidemiologic studies have revealed a clear link between disruption of circadian rhythms and cancer development in humans. Mice lacking the circadian genes Period1 and 2 (Per) or Cryptochrome1 and 2 (Cry) are deficient in cell cycle regulation and Per2 mutant mice are cancer-prone. However, it remains unclear how circadian rhythm in cell proliferation is generated in vivo and why disruption of circadian rhythm may lead to tumorigenesis.Methodology/Principal FindingsMice lacking Per1 and 2, Cry1 and 2, or one copy of Bmal1, all show increased spontaneous and radiation-induced tumor development. The neoplastic growth of Per-mutant somatic cells is not controlled cell-autonomously but is dependent upon extracellular mitogenic signals. Among the circadian output pathways, the rhythmic sympathetic signaling plays a key role in the central-peripheral timing mechanism that simultaneously activates the cell cycle clock via AP1-controlled Myc induction and p53 via peripheral clock-controlled ATM activation. Jet-lag promptly desynchronizes the central clock-SNS-peripheral clock axis, abolishes the peripheral clock-dependent ATM activation, and activates myc oncogenic potential, leading to tumor development in the same organ systems in wild-type and circadian gene-mutant mice.Conclusions/SignificanceTumor suppression in vivo is a clock-controlled physiological function. The central circadian clock paces extracellular mitogenic signals that drive peripheral clock-controlled expression of key cell cycle and tumor suppressor genes to generate a circadian rhythm in cell proliferation. Frequent disruption of circadian rhythm is an important tumor promoting factor.

CD34 antigen: Determination of specific sites of phosphorylation in vitro and in vivo

CD34, a type I transmembrane glycoprotein, is a surface antigen which is expressed on several cell types, including hematopoietic progenitors, endothelial cells, as well as mast cells. Recently, CD34 has been described as a marker for epidermal stem cells in mouse hair follicles, and is expressed in outer root sheath cells of the human hair follicle. Although the biological function and regulation of CD34 is not well understood, it is thought to be involved in cell adhesion as well as possibly having a role in signal transduction. In addition, CD34 was shown to be critical for skin tumor development in mice, although the exact mechanism remains unknown. Many proteins’ functions and biological activities are regulated through post-translational modifications. The extracellular domain of CD34 is heavily glycosylated but the role of these glycans in CD34 function is unknown. Additionally, two sites of tyrosine phosphorylation have been reported on human CD34 and it is known that CD34 is phosphorylated, at least in part, by protein kinase C; however, the precise location of the sites of phosphorylation has not been reported. In an effort to identify specific phosphorylation sites in CD34 and delineate the possible role of protein kinase C, we undertook the identification of the in vitro sites of phosphorylation on the intracellular domain of mouse CD34 (aa 309–382) following PKC treatment. For this work, we are using a combination of enzymatic proteolysis and peptide sequencing by mass spectrometry. After which the in vivo sites of phosphorylation of full-length mouse CD34 expressed from HEK293F cells were determined. The observed in vivo sites of phosphorylation, however, are not consensus PKC sites, but our data indicate that one of these sites may possibly be phosphorylated by AKT2. These results suggest that other kinases, as well as PKC, may have important signaling functions in CD34.

Evaluation of the Anti-inflammatory and Anti-tumor Effect of Ipomoea obscura (L) and Its Mode of Action Through the Inhibition of Pro Inflammatory Cytokines, Nitric Oxide and COX-2

Ipomoea obscura (L) is a widely used medicinal plant. In this study, we investigated its anti-inflammatory and anti-tumor effect using in vitro and in vivo models. Methanolic extract of I. obsucra (10 mg/kg b.wt) was given interaperitoneally before inducing inflammation (both acute and chronic) and tumor to mice. I. obscura produced significant inhibition of 55.6%, 42%, and 65% in the paw edema of animals induced by carrageenan, dextran, and formalin respectively. The extract was also a potent inhibitor of lipopolysaccharide (LPS)-induced NO, CRP, and proinflammatory cytokine production via gene expression in peritoneal macrophages. TNF-α production by macrophage culture treated with LPS was found to be significantly inhibited by I. obscura. The extract was 100% toxic at a concentration of 500 µg/mL for both Dalton's lymphoma ascites (DLA) and Ehrlich ascites carcinoma (EAC) cells. The extract was also found to inhibit tumor cell proliferation in a dose and time-dependent manner. It could also inhibit solid tumor development in mice induced with DLA cells and increased life span of mice bearing EAC tumor to 83% and 53.8%, respectively. This anti-inflammatory effect of the extract is assumed to result mainly from the inhibition of some key enzymes and mediators involved in the inflammation and/or cell signaling pathways such as iNOS, COX-2, and proinflammatory cytokines. This anti-inflammatory property might be the reason for its anti-tumor effects.

Abstract 1368: L-5F, an ApoA-I mimetic peptide, inhibits tumor angiogenesis by suppressing VEGF/bFGF signaling pathways

Abstract Objective We recently demonstrated that overexpression of ApoA-I inhibits tumor growth and improves survival in mice. In the present study we examined whether apoA-I mimetic peptide, L-5F reduces tumor development in mice by inhibiting angiogenesis. Methods Flank tumors were generated in immunocompetent C57BL6/J mice as described previously (Zhang L, et al. 2002) by injecting ID8 cells, a mouse ovarian cancer cell line. L-5F was injected s.c. once a day for 5 weeks (10 mg/kg). 3D vessel images of tumor tissues were created using a micro-CT scanner from Numira Biosciences. The effects of L-5F in vitro were studied in human umbilical vascular endothelial cells (HUVEC) following VEGF and bFGF treatment. MTS assay, BrdU cell proliferation assay, wound-healing assay, and trans-well assay were used to determine cell viability, proliferation, migration, and invasion of HUVEC, respectively. Activation of Erk and Akt pathways were analyzed using western blots. Results Daily s.c. injection of L-5F significantly reduced tumor size and quantity and size of vessels of flank tumors in C57BL6/J mice, compared to mice that did not receive L-5F peptide. L-5F (10 µg/ml) significantly inhibited both VEGF- and bFGF-induced cell viability (∼46% for VEGF and ∼56% for bFGF), proliferation (∼50% for VEGF and ∼70% for bFGF), migration (∼80% for VEGF and ∼77% for bFGF), and invasion (∼65% for VEGF and ∼44% for bFGF) in HUVECs. L-5F mediated effects were dose-dependent between 1 and 10 µg/ml including the inhibition VEGF and bFGF-induced phosphorylation of Erk and Akt. L-5F did not affect the viability and proliferation of HUVECs in the basal state (i.e. absence of VEGF and bFGF treatments). Conclusion Our data suggest that apoA-I mimetic peptide, L-5F inhibits tumor development in mice, in part, by inhibiting angiogenesis. ApoA-I mimetic peptides may serve as novel anti-angiogenesis and anticancer therapies. 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 1368.

Abstract 5324: Ablation of IL-17A abrogates progression of spontaneous intestinal tumorigenesis

Abstract The intestinal tract is subject to constant stimulation of immune cells and intestinal epithelial cells either by noninfectious antigens or infection. Recent studies identified the role of IL-17A in chronic inflammation in intestinal epithelium. Yet the intrinsic role of endogenous IL-17A in spontaneous intestinal tumorigenesis has not been addressed previously. Ablation of IL-17A significantly reduced tumor development in mice bearing a heterozygote mutation in the APC (adenomatous polyposis coli) gene (ApcMin/+ mice). There was a marked decrease in IL-6 and IL-23 (p19), as well as a decrease in IL-1β, suggesting that the inflammatory microenvironment was significantly changed by ablating IL-17A. We also found a significant decrease in CD44 and proinflammatory mediators such as KC (Keratinocyte Chemoattractant) and Cox-2. There was also a reduced infiltration of lymphocytes including T cells and preservation of intestinal architecture and the presence of APC protein in intestinal epithelial cells in IL-17AKO ApcMin/+ mice. Interestingly, IL-17A ablation also corrected immunological abnormalities such as splenomegaly and thymic atrophy in ApcMin/+ mice. We tested whether CD4 T cells from ApcMin/+ mice are more proliferative based on its role as a tumor suppressor gene, thereby regulating cell cycle. We observed that CD4 T cells from ApcMin/+ mice were hyperproliferative when compared with their wildtype littermate controls upon TcR (T cell receptor) stimulation. This hyperproliferation was more obvious following stronger stimulation through the TcR. Next, we tested whether the effector CD4 T cells are more refractory to regulatory T cell (Treg)-mediated suppression. Upon co-culture with wildtype Treg cells, ApcMin/+ CD4 effector T cells were more resistant to Treg-mediated suppression especially at a high ratio of effectors to Treg, suggesting that the suppression on ApcMin/+ CD4 effector T cells are not as efficient as for wildtype CD4 effector T cells. Finally, these CD4 T cells from ApcMin/+ mice induced colitis in immunodeficient mice upon adoptive transfer, while the ablation of IL-17A in CD4 T cells in ApcMin/+ mice (IL-17A KO ApcMin/+ mice) completely abolished this pathogenic potential in vivo, clearly showing that ablation of IL-17A abrogated hyperproliferation and colitogenic potential in immunodeficient mice. This was consistent with our observation of the phenotypic changes in intestinal tumorigenesis. Taken together, our results suggest an important proinflammatory role of IL-17A and its correction of immune abnormalities in spontaneous intestinal tumorigenesis. We demonstrate altered CD4 T cell functions from ApcMin/+ mice which favors intestinal tumorigenesis and the ablation of IL-17A can abolish these altered function, suggesting the importance of T-cell mediated IL-17A in spontaneous intestinal tumorigenesis 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 5324.

Abstract 1602: Deletion of cyclooxygenase-2 in mouse mammary epithelial cells delays breast cancer onset

Abstract Cyclooxygenase 2 (COX-2) enzyme, which catalyzes the synthesis of several pro- and anti-tumorigenic prostanoids, is associated with more than 40% of breast cancers. Selective and non-selective COX-2 inhibitors were shown to decrease the risk of tumorigenesis and breast cancer recurrence. However, the precise role of COX-2 in the mammary epithelium, and the potential role of COX-1, remains ill-defined. This is in part because of infertility, renal pathology and short life-span that accompanies global deletion of the COX-2 gene in mice. To study the role and mechanisms of COX-2 actions in breast cancer, we engineered mice on a pure FVB/N background that lack COX-2 only in mammary epithelial cells (MECs). Mice transgenic for COX-2 exons 5 and 6, flanked by loxP sites (COX-2flox/flox), were crossed with mice expressing cre recombinase under control of the mouse mammary tumor virus (mmtv) promoter (Cremmtv) to generated the MEC-targeted deletion of COX-2. MECs harvested from wild type (WT; COX-2flox/flox) and knock-out (KO; Cremmtv COX-2flox/flox) mice were treated, in vitro, with or without a single dose of 5µg/ml lipopolysaccaride (LPS). KO MECs showed about 80% less COX-2 mRNA and protein expression. Prostaglandin (PG) E2 production was similarly reduced in KO MECs, despite unchanged levels of COX-1 mRNA and protein (n=5-7). We confirmed COX-2 as the principle source of PGE2 in MECs using inhibitors specific for COX-2 (rofecoxib) or COX-1 (FR122047) (n=6). Expression of COX-2 and COX-1 mRNA and protein, and PGE2 generation, were not significantly different in untreated or LPS-treated peritoneal macrophages obtained from WT and KO mice (n=5-6) confirming the specificity of the targeted COX-2 deletion to MECs. Unlike global COX-2 deficiency, mice lacking COX-2 in MECs were viable, fertile and disease-free making them highly suitable for cancer studies. To induce breast tumorigenesis, 6 week old virgin female WT and KO mice received medroxyprogesterone acetate implants (75 mg/pellet, 21 day release) subcutaneously followed, at 9 weeks of age, by 1mg/week of 7,12-dimethylbenz[a]anthracene (DMBA) administration for 4 weeks. This regime causes predominantly mammary tumor development in mice. Starting from week 13, mice were checked weekly for mammary tumors. The onset of breast cancer, denoted as the week of age during which a mammary tumor was palpated in the glands of a live mouse, was significantly delayed in KO mice compared to their WT counterparts (p=0.03); the median duration of tumor free period was 28 weeks for KO animals (n=36) versus 22 weeks for WT mice (n=20). These data indicate that targeted deletion of COX-2 in mammary epithelium affords significant protection against initiation and progression of breast tumorigenesis. 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 1602.

Abstract 4210: Gsta4 modifies susceptibility to skin tumor development in mice and humans

Abstract The incidence of non-melanoma skin cancer (NMSC) is equivalent to that of all other human cancers combined, and genetic factors contribute to risk of the disease. Using genetic crosses of skin tumor promotion sensitive DBA/2 mice with relatively resistant C57BL/6 mice, loci that modify susceptibility to tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) have been mapped to chromosomes (chr) 1, 2, 9, and 19. Here, we show that Glutathione S-transferase alpha 4 (Gsta4), which maps to skin tumor promotion susceptibility locus Psl1.2 on chr 9, was expressed at significantly different levels in the epidermis of C57BL/6 versus DBA/2 mice following treatment with diverse promoting agents. Gsta4 expression was dramatically upregulated in the epidermis of C57BL/6 mice, but not DBA/2 mice, following treatment with TPA, okadaic acid, chrysarobin, or ultraviolet light. Gsta4 deficient mice were more susceptible to skin tumor development in the two-stage skin carcinogenesis protocol providing compelling evidence that Gsta4 underlies the effect of Psl1.2 on susceptibility to skin tumor promotion by TPA. A number of polymorphisms were detected in the putative promoter region of Gsta4 in C57BL/6 versus DBA/2 mice, and ongoing studies are addressing the genetic basis of strain-specific expression of Gsta4 during tumor promotion. Finally, inheritance of polymorphisms in GSTA4 was associated with risk of NMSC in human populations, further supporting Gsta4/GSTA4 as a gene that modifies susceptibility to skin tumor development. Supported by NIH grant ES016623. 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 4210.

Abstract 1871: Honokiol, a natural plant product, inhibits ultraviolet radiation-induced skin tumorigenesis in mice through inhibition of inflammation and inflammatory mediators

Abstract Chronic exposure of solar ultraviolet (UV) radiation is the major etiologic agent for over one million new cases of non-melanoma skin cancers in the US each year. These cutaneous malignancies thus have a tremendous impact on public health and healthcare expenditures. The use of natural plant products has received considerable interest to protect skin from adverse biological effects of UV radiation since the protective clothing and use of sunscreens are not adequate for skin photoprotection. The photoprotective effect of honokiol, a plant product from Magnolia species, therefore assessed using SKH-1 hairless mouse model. To determine the skin cancer chemopreventive effect and associated mechanism of honokiol, a hydrophilic cream based topical formulation was developed. Different groups of mice (n=20) treated with or without honokiol were subjected to photocarcinogenesis protocol. Mice were irradiated to UVB (180 mJ/cm2) three times a week. At the termination of the experiment at 24th week, we found that topical application of honokiol (1mg &amp; 3mg/mouse) inhibited UVB-induced skin tumor development in terms of tumor incidence (10-20%), tumor multiplicity (40-60%) and tumor size (50-75%). Since UV-induced inflammation plays a crucial role in the development of skin tumors, we examined the effect of honokiol on UVB-induced biomarkers of inflammation, such as the expression levels of cyclooxygenase-2 (COX-2), prostaglandins (PG), proliferating cell nuclear antigen (PCNA), infiltration of inflammatory leukocytes, development of edema and epidermal hyperplastic response using immunostaining, western blotting and RT-PCR. Our data revealed that topical application of honokiol before each exposure of UVB radiation resulted in a dose-dependent inhibition of UVB-induced expression of COX-2, PGE2, developments of edema and infiltration of leukocytes at the UVB-irradiated skin sites. Honokiol also inhibited the levels of PCNA (hyperplastic potential) in epidermal keratinocytes of UVB-exposed skin. The inhibitory effects of honokiol on inflammatory mediators were also found in skin tumor samples. The results of this study suggest that honokiol has the ability to inhibit UVB radiation-induced skin tumor development in mice and it is associated with the inhibition of UVB-induced inflammation and inflammatory mediators in mouse skin and skin tumors. 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 1871.