Reduced Tumor Multiplicity Research Articles

Electrochemically Reduced Water Delays Mammary Tumors Growth in Mice and Inhibits Breast Cancer Cells Survival In Vitro.

Electrochemical reduced water (ERW) has been proposed to have beneficial effects on human health due to its rich content of H2 and the presence of platinum nanoparticles with antioxidant effects. Many studies have demonstrated that ERW scavenging properties are able to reduce the damage caused by oxidative stress in different experimental models. Although few in vivo studies have been reported, it has been demonstrated that ERW may display anticancer effects by induction of tumor cells apoptosis and reduction of both angiogenesis and inflammation. In this study, we show that ERW treatment of MCF-7, MDA-MB-453, and mouse (TUBO) breast cancer cells inhibited cell survival in a time-dependent fashion. ERW decreased ErbB2/neu expression and impaired pERK1/ERK2 and AKT phosphorylation in breast cancer cells. In addition, ERW treatment induced apoptosis of breast cancer cell lines independently of the status of p53 and ER and PR receptors. Our in vivo results showed that ERW treatment of transgenic BALB-neuT mice delayed the development of mammary tumors compared to the control. In addition, ERW induced a significant prolongation of tumor-free survival and a reduction in tumor multiplicity. Overall, these results suggest a potential beneficial role of ERW in inhibiting cancer cells growth.

Abstract 5267: Chemoprevention of lung carcinogenesis by dietary nicotinamide and inhaled budesonide

Abstract Nicotinamide, the amide form of vitamin B3, and budesonide, a synthetic glucocorticoid used in the treatment of asthma, were evaluated for efficacy of chemoprevention of lung adenoma formation in a benzo(a)pyrene murine carcinogenesis model. Female A/J mice were given three doses of 3 mg benzo[a]pyrene three days apart and started on experimental diets either 1 week (early stage intervention) or 8 weeks (late stage) after last carcinogen dose. Aerosol treatments were delivered 5 days per week starting 1 or 8 weeks post carcinogen. Nicotinamide, fed at a dietary concentration of 0.75% starting 1 week after last carcinogen administration, inhibited adenoma formation by 54%, and by 39% when begun 8 weeks post final carcinogen. Nicotinamide fed at 0.25% of the diet was not found to effect adenoma formation . Dietary nicotinamide under the same conditions with budesonide administered via aerosol administration at 25μg/kg, reduced lung adenoma formation by 90% at early stage intervention and 49% at late stage intervention. Budesonide alone administered at the same dose level gave 77% inhibition at early stage and 41% inhibition at late stage. Decreases in adenoma formation were statistically significant via ANOVA analysis at both early and late interventions. Combination therapy decreased adenoma formation significantly over either individual therapy at the early stage and significantly compared to nicotinamide alone at the late stage. However, administration of nicotinamide by aerosol inhalation at doses up to 15 mg/kg/day did not result in a statistically significant reduction in tumor multiplicity at 1 or 8 weeks post last carcinogen administration . There is a high level of clinical interest in minimal toxicity lung cancer chemoprevention strategies. We feel administration of a safe dietary agent such as nicotinamide when combined with direct epithelial delivery of a glucocorticoid such as budesonide, commonly used for asthma therapy, is a promising approach for aerodigestive chemoprevention. Citation Format: Donna Seabloom, Beverly Wuertz, Art Galbraith, Jenny Antonides, Vernon Steele, Lee Wattenberg, Frank G. Ondrey. Chemoprevention of lung carcinogenesis by dietary nicotinamide and inhaled budesonide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5267. doi:10.1158/1538-7445.AM2017-5267

Stress Response Protein RBM3 Promotes the Development of Colitis-associated Cancer.

Colitis-associated cancer (CAC) is caused by chronic intestinal inflammation and often results from refractory inflammatory bowel disease (IBD). Stress response proteins Cirp and HSPA4 are involved in the refractory clinical course and development of CAC. RNA-binding motif protein 3 (RBM3) is induced in response to various stresses and is upregulated in several cancers. However, the role of RBM3 in CAC is unclear. We assessed RBM3 expression and function in 263 human intestinal mucosa samples from patients with IBD and in Rbm3-deficient (Rbm3) mice. Expression of RBM3 was correlated with the expression of stress response proteins Cirp, HSPA4, and HSP27 in the colonic mucosa of patients with IBD. Significant correlation was observed between the expression of RBM3 and that of Bcl-xL or stem cell markers. RBM3 expression increased and significantly correlated with R-spondin expression in the colonic mucosa of patients with refractory IBD, a condition associated with increased cancer risk, and RBM3 was overexpressed in human CACs. In the murine CAC model, Rbm3 deficiency decreased R-spondin and Bcl-xL expression and increased apoptotic cell number in the colonic mucosa, leading to reduced tumor multiplicity. Transplantation of wild-type and Rbm3 bone marrow did not alter tumor burden, indicating the importance of RBM3 in epithelial cells. Our findings indicated that RBM3 was required for efficient inflammatory carcinogenesis in the murine CAC model and suggested that RBM3 could be a predictive biomarker of CAC risk and a new therapeutic target for cancer prevention in patients with IBD.

Anti-inflammatory natural product goniothalamin reduces colitis-associated and sporadic colorectal tumorigenesis.

The tumor microenvironment offers multiple targets for cancer therapy, including pro-tumorigenic inflammation. Natural compounds represent an enormous source of new anti-inflammatory and anticancer agents. We previously showed that the styryl lactone goniothalamin (GTN) has promising antiproliferative and anti-inflammatory activities. Because inflammation is a major driver of colorectal cancer (CRC), we therefore evaluated the therapeutic and preventive potentials of GTN in colitis, colitis-associated cancer (CAC) and spontaneous CRC. First, in a simplistic model of inflammation in vitro, GTN was able to inhibit cytokine production in bone marrow-derived macrophages induced by lipopolysaccharide. Next, in dextran sulfate sodium (DSS) induced-colitis model, mice treated with GTN displayed restored tissue architecture, increased cell proliferation in the colonic crypts and reduced epithelial damage. Moreover, colon tissue from GTN-treated mice had significantly less expression of the inflammatory genes interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), S100A9, interleukin 23A (IL-23A), IL-22 and IL-17A In the azoxymethane/DSS model of CAC, GTN reduced tumor multiplicity, load and size. Additionally, GTN suppressed production of IL-6, IL-17 and TNF-α in tumor tissue, as well as abrogated stromal immune cell activation and nuclear translocation of NF-κB. Finally, in a tamoxifen inducible model of sporadic CRC, GTN-treated mice had significantly fewer tumors and decreased levels of IL-17A, IL-6, S100A9 and TNF-α protein within the tumors. These results suggest that GTN possesses anti-inflammatory and antitumor activities and represents a preventive and therapeutic agent modulating the inflammatory environment in the colon during colitis as well as CAC and CRC development.

Abstract 748: Targeting the crosstalk between tumor cells and adipocytes to block breast cancer progression

Abstract It is now well-established that the local microenvironment of an emerging tumor plays a vital role in various steps of tumorigenesis. Stromal cells, such as fibroblasts, endothelial and various inflammatory cells, can contribute to tumor progression, and tumor cells can directly model their microenvironment, influencing host cells to secrete factors that favor cancer cell survival and growth. Recent studies have demonstrated the existence of cross-talk between breast cancer (BC) cells and adipocytes, the most abundant stromal cell in the breast, supporting their involvement in BC and consistent with epidemiological studies reporting the association of obesity and insulin resistance/diabetes with a higher risk and poor prognosis of BC. Based on this evidence, we investigated whether adipocyte de-differentiation is the crucial trigger in inducing pro-tumor microenvironment remodeling and tumor progression. Co-culture of 4T1 mouse mammary tumor cells using Transwell or their conditioned medium with mature adipocytes derived from the 3T3-L1 mouse cells induced adipocyte de-differentiation, observed as a reduced expression of specific differentiation markers (peripilin, adipocyte protein 2 (aP2), PPARã and adiponectin) in Western blots and by qRT-PCR, and a reduced triglyceride content as assessed by oil red O staining compared to mature adipocytes. Strong adipocyte de-differentiation, associated with an increase in pro-inflammatory molecules (IL6 and CCL2), was also induced in vitro by tumor interstitial fluids obtained from MMT-PyMT transgenic mice, but not by plasma of these mice. Preliminary studies to identify molecules involved in the cross-talk indicated that the phenomenon is exosome-unrelated. Based on the relevant role of the PPARã transcription factor in inducing and maintaining adipocyte differentiation, we co-cultured adipocytes with 4T1 BC cells in the presence of several PPARã agonists enriched in the diets of many populations displaying a low incidence of cancer (þ3-fatty acids), and in the presence of glitazones (rosiglitazone and pioglitazone) to force adipocyte stable differentiation. While most of these compounds did not block adipocyte de-differentiation induced by tumor cells, pioglitazone and eicosanoid acid showed some activity in inhibiting this de-differentiation in vitro. Treatment of MMTV-PyMT transgenic mice at tumor onset by oral gavage with pioglitazone did not significantly reduce mammary tumor growth compared to controls, whereas eicosanoid acid treatment significantly reduced tumor multiplicity and tumor burden compared to controls. Overall, our results suggest that tumor-induced adipocyte de-differentiation participates in tumorigenesis and that the blockade of this program can inhibit tumor progression. Supported by AIRC and Fondazione Cariplo. Citation Format: Tiziana Triulzi, Valentina Ciravolo, Viola Regondi, Martina Di Modica, Claudia Chiodoni, Elda Tagliabue. Targeting the crosstalk between tumor cells and adipocytes to block breast cancer progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 748.

Deguelin Analogue SH-1242 Inhibits Hsp90 Activity and Exerts Potent Anticancer Efficacy with Limited Neurotoxicity.

The Hsp90 facilitates proper folding of signaling proteins associated with cancer progression, gaining attention as a target for therapeutic intervention. The natural rotenoid deguelin was identified as an Hsp90 inhibitor, but concerns about neurotoxicity have limited prospects for clinical development. In this study, we report progress on deguelin analogues that address this limitation, focusing on the novel analogue SH-1242 as a candidate to broadly target human lung cancer cells, including those that are chemoresistant or harboring KRAS mutations. In a KRAS-driven mouse model of lung cancer, SH-1242 administration reduced tumor multiplicity, volume, and load. Similarly, in human cell line-based or patient-derived tumor xenograft models, SH-1242 induced apoptosis and reduced tumor vasculature in the absence of detectable toxicity. In contrast to deguelin, SH-1242 toxicity was greatly reduced in normal cells and when administered to rats did not produce obvious histopathologic features in the brain. Mechanistic studies revealed that SH-1242 bound to the C-terminal ATP-binding pocket of Hsp90, disrupting the ability to interact with its co-chaperones and clients and triggering a degradation of client proteins without affecting Hsp70 expression. Taken together, our findings illustrate the superior properties of SH-1242 as an Hsp90 inhibitor and as an effective antitumor and minimally toxic agent, providing a foundation for advancing further preclinical and clinical studies.

PAK1 promotes intestinal tumor initiation.

p21-activated kinase 1 (PAK1) is a serine/threonine kinase that is overexpressed in colorectal cancer. PAK1 is a target of mesalamine [5-aminosylicylic acid (5-ASA)], a common drug for the treatment of ulcerative colitis with prospective chemopreventive properties. Here, we investigated whether PAK1 deletion impedes tumorigenesis in murine intestinal cancer models. Ten-week-old APC(min) or APC(min)/PAK1(-/-) mice were monitored for 8 weeks, euthanized, and assessed for tumor number and size. Six- to 8-week-old PAK1(-/-) and wild-type (WT) mice received one 10 mg/kg intraperitoneal injection of azoxymethane (AOM) and four cycles of 1.7% dextran sodium sulfate (DSS) for 4 days followed by 14 days of regular water. Mice also received 5-ASA via diet. Tumor incidence and size was assessed via colonoscopy and pathology. Molecular targets of PAK1 and 5-ASA were evaluated via immunohistochemistry (IHC) in both models. PAK1 deletion reduced tumor multiplicity and tumor burden but did not alter average tumor size in APC(min) mice. IHC revealed that PAK1 deletion reduced p-AKT, β-catenin, and c-Myc expression in APC(min) adenomas. Colonoscopy and pathologic analysis revealed that PAK1 deletion reduced tumor multiplicity without affecting tumor size in AOM/DSS-treated mice. 5-ASA treatment and PAK1 deletion impeded tumor multiplicity and dysplastic lesions in AOM/DSS mice. IHC further revealed that 5-ASA blocked β-catenin signaling via inhibition of PAK1/p-AKT. These data indicate that PAK1 contributes to initiation of intestinal carcinogenesis.

Abstract 893: Metabolomic profiling of APCΔ14/+ mice maintained on a methyl donor deficient diet reveals alterations to methionine and fatty acid metabolism associated with cancer protection

Abstract Our laboratory has previously demonstrated that administration of a diet deficient in methyl donor nutrients (folic acid, methionine, choline and vitamin B12) reduces tumor multiplicity by greater than 95% in ApcMin/+ mice. However, the mechanism by which this dramatic cancer protection is afforded is unknown. To begin to identify the metabolic consequences of methyl donor deficiency, we have conducted an LC/MS-based metabolic profiling analysis of colonic mucosa collected from ApcΔ14/+ mice fed a methyl donor deficient (MDD) diet for 17 weeks. Methyl donor deficiency causes significant alterations to a number of key metabolic pathways, including those involved in the methionine cycle, fatty acid oxidation, and tryptophan catabolism. We observed significant reductions in methionine (65% reduction, p<0.001) and betaine (70% reduction, p<0.001), accompanied by large increases in homocysteine (88.6-fold increase, p<0.001) and S-adenosylmethionine (17.2-fold increase, p<0.001), confirming that dietary methyl donor deficiency alters one-carbon metabolites in the colon. MDD mice were found to have reduced levels of acyl-carnitines and free carnitine (58% reduction, p<0.001). Additionally, we detected a reduction in the critical metabolic component, acetyl CoA, suggesting decreased transport of fatty acids into the mitochondria for use in fatty acid oxidation (FAO). Finally, we observed changes in the mucosal levels of tryptophan (32% reduction, p = 0.02), kynurenine (2.8-fold increase, p = 0.008) and indolepropionate (5.1-fold increase, p = 0.005), that were retained for seven weeks beyond active methyl donor deficiency. Taken together, these findings suggest that dietary methyl donor deficiency significantly disrupts the methionine cycle, inhibits fatty acid transport and oxidation, and may induce long-term changes to tryptophan metabolism. This study has improved our understanding of the molecular mechanisms underlying the cancer protection conveyed by dietary methyl donor deficiency and has identified metabolic pathways that are candidate targets for chemopreventive intervention. Citation Format: Matthew P. Hanley, Daniel W. Rosenberg. Metabolomic profiling of APCΔ14/+ mice maintained on a methyl donor deficient diet reveals alterations to methionine and fatty acid metabolism associated with cancer protection. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 893. doi:10.1158/1538-7445.AM2015-893

Abstract 896: Correlation between modulation of RNA expression and the preventive and therapeutic efficacy of Tamoxifen in an ER+ model of breast cancer

Abstract The SERMs, primarily Tamoxifen, have been employed in both the therapy and prevention of ER+ breast cancer for many decades. In the present study we examined the effects of the SERM tamoxifen as a preventive and therapeutic agent in an ER+ Methylnitrosourea (MNU) -induced model of mammary cancer in rats. Fifty day old female Sprague Dawley rats were administered MNU i.v. and tamoxifen was administered continually in diet beginning either 5 days post MNU (prevention) or when an animal developed its first palpable tumor (150mm2) [therapy]. The approximate ED50 (dose required to reduce tumor multiplicity 50%) and ED90 in the preventive assays were 0.66 and 3.3 ppm (mg of tamoxifen per g of diet) for Tamoxifen. Interestingly the 3.3 ppm dose was ineffective as a therapy at these doses although it slowed growth of palpable lesions. Rather one had to administer a dose of roughly 100 ppm achieve a strong therapeutic response. We also examined the short term effects (5 days of treatment) of 3.3 and 100 ppm on gene expression in palpable lesions. We compared each treatment group with the control to find the up or down regulated genes. The number of differentially expressed (DE) genes selected at FDR = 0.2 increased from 584 to 2267 when the dose increased from 3.3ppm to 100ppm. The 3.3ppm treatment yielded fewer DE genes compared to the 100ppm treatment, but the majority (60%) of the genes identified in the 3.3 ppm group were identified in the high dose group. The higher dose profoundly decreased a variety of specific genes in the proliferative pathways, particularly in APC (Anaphase Prometaphase Condensation Complex), which includes Ccnb1 and Top2A. In contrast, the low dose failed to significantly change expression of these specific genes. The effects of the higher dose of tamoxifen were confirmed by the use of RT-PCR. Using the human Tamoxifen treatment dataset GSE6532 in GEO, we found 633 DE genes by comparing the treated (n = 277) and untreated (n = 237) patients. We found 68 genes shared between human and rat derived from 100ppm treatment and 11 genes from 3.3ppm treatment. 9 of the 11 genes regulated by the low dose were also regulated by the high dose. These genes included RAB5B, a member of the RAS oncogene family, and GDF9, the growth differentiation factor 9. By gene enrichment analysis (GSEA), we found several pathways, including RAS, TGF beta signaling, DNA damage signaling, and cell cycle pathways, which were affected by the tamoxifen treatment. Our study suggests that the rat gene expression was regulated by tamoxifen even at low dose, and the DE genes at the low dose are a subset of these DE genes altered at the high dose. These data provide a rationale for testing low dose tamoxifen as a chemo preventive agent for women at high risk of developing breast cancer and offer potential genomic biomarkers associated with efficacy at these lower doses. Citation Format: Howard H. Yang, Huaitian Liu, Ronald Lubet, Clinton J. Grubbs, Holly Nicastro, Maxwell P. Lee. Correlation between modulation of RNA expression and the preventive and therapeutic efficacy of Tamoxifen in an ER+ model of breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 896. doi:10.1158/1538-7445.AM2015-896

Enhanced Percutaneous Delivery of 1,1-bis(3'-indolyl)-1-(p-chlorophenyl) Methane for Skin Cancer Chemoprevention.

Skin cancer has high incidence in the United States and is mainly caused by ultraviolet B (UVB) radiation. In this study, we demonstrated the role of 1,1-bis(3'-indolyl)-1-(p-chlorophenyl) methane (DIM-D) in the prevention of skin photocarcinogenesis using an in vivo UVB-induced skin cancer model. We also evaluated the efficiency of oleic acid-modified nanostructured lipid carriers to deliver DIM-D across the skin barrier into the epidermis for chemopreventive activity. Nanocarriers were 203.00 ± 21.21 nm in diameter with polydispersity, zeta potential and entrapment efficiency of 0.33 ± 0.01, 37.17 ± 0.90 mV and 93.64 ± 0.65%, respectively. Oleic acid-modified nanocarriers were incorporated into Hydroxypropyl methylcellulose to form DIM-D-Nanogel (DIM-D-N). DIM-D-N pretreatment prior to UVB exposure delayed tumor initiation and reduced tumor multiplicity (p < 0.05) at the end of the study compared to Epigallocatechin gallate (EGCG) gel pretreatment. DIM-D-N pretreatment decreased UVB-induced damage to skin lipids and proteins (p < 0.05), respectively by 7.63 and 2.56-fold less than EGCG gel pretreatment and by 17.86 and 11.92-fold less than UVB-only treatment. Histology showed rete-ridge extension, epidermal thickening and hyperkeratosis for UVB-only treatment and EGCG gel pretreatment; DIM-D-N pretreatment showed similar features as the negative control. Western blot analysis showed increased Nurr1 expression (p < 0.05) for DIM-D-N pretreated group compared to EGCG gel (4.68-fold). DIM-D-N pretreatment reduced BCI-2 expression (p < 0.05) but increased Bax and cPARP. Knock down studies with Nurr1 siRNA reduced the expressions of Nurr1 and cPARP by 8.18 and 1.45-fold, respectively (p < 0.05). Our results suggest the role of DIM-D in skin cancer chemoprevention mediated by possible molecular therapeutic targets such as Nurr1.

Abstract 3667: Attenuation of NF-κB signaling in myeloid cells enhances urethane-induced lung tumorigenesis via neutrophil-derived IL-1β

Abstract The transcription factor Nuclear Factor κB (NF-κB) is a master regulator of inflammation, and its activation is associated with tumorigenesis in a variety of cancers. Our group and others have shown that NF-κB signaling in lung epithelial cells is critical for lung carcinogenesis. However, pharmaceutical inhibition of NF-κB signaling has not been effective for the treatment of lung tumorigenesis in animals or humans for reasons that remain unknown. We hypothesized that attenuation of NF-κB signaling in myeloid cells contributes to inefficacy of NF-κB-targeted therapies by unmasking pro-tumorigenic characteristics of inflammatory cells. For our studies, we utilized mice with IKKβ deleted specifically in myeloid cells (IKKβΔmye) and induced lung tumorigenesis with a single intraperitoneal injection of the lung carcinogen urethane (1g/kg). We found that IKKβΔmye mice developed more atypical adenomatous hyperplasia (AAH) lesions at 6 weeks and more lung tumors at 16 weeks after urethane compared to WT mice. These histological differences were correlated with enhanced lung inflammation in IKKβΔmye mice which was observed 1 week after urethane and persisted for up to 6 weeks. Flow cytometric analysis of myeloid cell populations revealed that IKKβΔmye mice contained more pulmonary neutrophils at 1 and 6 weeks after urethane compared to WT mice. In bone marrow chimeras generated by transplantation of bone marrow from IKKβΔmye or WT donors into recipient NF-κB reporter mice, we showed that depletion of neutrophils using Ly6G antibody treatment (100 ug/twice weekly IP injection) during the first 6 weeks of lung carcinogenesis blocked NF-κB activation in stromal cells and reduced tumor multiplicity in IKKβΔmye mice to levels observed in urethane-treated WT mice. Thus, these studies indicated that neutrophils play an important role in lung tumorigenesis during early tumor development and may do so by limiting NF-κB activation in lung epithelial cells. Evaluation of cytokines in the lungs of WT and IKKβΔmye mice at 1 week after urethane revealed higher levels of IL-1β in IKKβΔmye mice compared to WT mice. Further examination of myeloid cells sorted 1 week after urethane identified neutrophils as the key producers of IL-1β in the lungs of IKKβΔmye mice. Neutralization of IL-1 signaling by IL-1 receptor antagonist (IL-1ra) treatment during the first 4 weeks of carcinogenesis decreased the number of AAH lesions in IKKβΔmye mice 6 weeks after urethane and reduced the number of tumors at 16 weeks in IKKβΔmye mice. Taken together, our data suggest that neutrophils can support tumor promotion through production and secretion of IL-1β, which may activate pro-tumorgenic NF-κB signaling in the lung epithelium. We speculate that the paradoxical increase in inflammation and IL-1β production resulting from NF-κB inhibition in myeloid cells contributes to the lack of effectiveness of NF-κB inhibitors in patients with lung cancer. Citation Format: Allyson McLoed, Rinat Zaynagetdinov, Taylor Sherrill, Fiona Yull, Timothy Blackwell. Attenuation of NF-κB signaling in myeloid cells enhances urethane-induced lung tumorigenesis via neutrophil-derived IL-1β. [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 3667. doi:10.1158/1538-7445.AM2014-3667

P63 is a prosurvival factor in the adult mammary gland during post-lactational involution, affecting PI-MECs and ErbB2 tumorigenesis

In embryogenesis, p63 is essential to develop mammary glands. In the adult mammary gland, p63 is highly expressed in the basal cell layer that comprises myoepithelial and interspersed stem/progenitor cells, and has limited expression in luminal epithelial cells. In adult skin, p63 has a crucial role in the maintenance of epithelial stem cells. However, it is unclear whether p63 also has an equivalent role as a stem/progenitor cell factor in adult mammary epithelium. We show that p63 is essential in vivo for the survival and maintenance of parity-identified mammary epithelial cells (PI-MECs), a pregnancy-induced heterogeneous population that survives post-lactational involution and contain multipotent progenitors that give rise to alveoli and ducts in subsequent pregnancies. p63+/- glands are normal in virgin, pregnant and lactating states. Importantly, however, during the apoptotic phase of post-lactational involution p63+/- glands show a threefold increase in epithelial cell death, concomitant with increased activation of the oncostatin M/Stat3 and p53 pro-apoptotic pathways, which are responsible for this phase. Thus, p63 is a physiologic antagonist of these pathways specifically in this regressive stage. After the restructuring phase when involution is complete, mammary glands of p63+/- mice again exhibit normal epithelial architecture by conventional histology. However, using Rosa(LSL-LacZ);WAP-Cre transgenics (LSL-LacZ, lox-stop-lox β-galactosidase), a genetic in vivo labeling system for PI-MECs, we find that p63+/- glands have a 30% reduction in the number of PI-MEC progenitors and their derivatives. Importantly, PI-MECs are also cellular targets of pregnancy-promoted ErbB2 tumorigenesis. Consistent with their PI-MEC pool reduction, one-time pregnant p63+/- ErbB2 mice are partially protected from breast tumorigenesis, exhibiting extended tumor-free and overall survival, and reduced tumor multiplicity compared with their p63+/+ ErbB2 littermates. Conversely, in virgin ErbB2 mice p63 heterozygosity provides no survival advantage. In sum, our data establish that p63 is an important survival factor for pregnancy-identified PI-MEC progenitors in breast tissue in vivo.

Lycopene Metabolite, Apo-10′-Lycopenoic Acid, Inhibits Diethylnitrosamine-Initiated, High Fat Diet–Promoted Hepatic Inflammation and Tumorigenesis in Mice

Obesity is associated with increased risk in hepatocellular carcinoma (HCC) development and mortality. An important disease control strategy is the prevention of obesity-related hepatic inflammation and tumorigenesis by dietary means. Here, we report that apo-10'-lycopenoic acid (APO10LA), a cleavage metabolite of lycopene at its 9',10'-double bond by carotene-9',10'-oxygenase, functions as an effective chemopreventative agent against hepatic tumorigenesis and inflammation. APO10LA treatment on human liver THLE-2 and HuH7 cells dose dependently inhibited cell growth and upregulated sirtuin 1 (SIRT1), a NAD(+)-dependent protein deacetylase that may suppress hepatic carcinogenesis. This observed SIRT1 induction was associated with decreased cyclin D1 protein, increased cyclin-dependent kinase inhibitor p21 protein expression, and induced apoptosis. APO10LA supplementation (10 mg/kg diet) for 24 weeks significantly reduced diethylnitrosamine-initiated, high fat diet (HFD)-promoted hepatic tumorigenesis (50% reduction in tumor multiplicity; 65% in volume) and lung tumor incidence (85% reduction) in C57Bl/6J mice. The chemopreventative effects of APO10LA were associated with increased hepatic SIRT1 protein and deacetylation of SIRT1 targets, as well as with decreased caspase-1 activation and SIRT1 protein cleavage. APO10LA supplementation in diet improved glucose intolerance and reduced hepatic inflammation [decreased inflammatory foci, TNFα, interleukin (IL)-6, NF-κB p65 protein expression, and STAT3 activation] in HFD-fed mice. Furthermore, APO10LA suppressed Akt activation, cyclin D1 gene, and protein expression and promoted PARP protein cleavage in transformed cells within liver tumors. Taken together, these data indicate that APO10LA can effectively inhibit HFD-promoted hepatic tumorigenesis by stimulating SIRT1 signaling while reducing hepatic inflammation.

Thymoquinone inhibits cell proliferation through regulation of G1/S phase cell cycle transition in N-nitrosodiethylamine-induced experimental rat hepatocellular carcinoma

Dysregulated cell proliferation and tumorigenesis is frequently encountered in several cancers including hepatocellular carcinogenesis (HCC). Thus, agents that inhibit cell proliferation and restrain hepatic tumorigenesis through cell cycle regulation have a beneficial effect in the treatment of hepatocellular carcinogenesis. The present study was aimed to investigate the efficacy of thymoquinone (TQ), an active compound derived from the medicinal plant Nigella sativa, on N-nitrosodiethylamine (NDEA) [0.01% in drinking water for 16 weeks]-induced hepatocarcinogenesis in experimental rats. After experimental period, the hepatic nodules, liver injury markers and tumor markers levels were substantially increased in NDEA induced liver tumors in rats. However, TQ (20mg/kg body weight) treatment greatly reduced liver injury markers and decreased tumor markers and prevented hepatic nodule formation and reduced tumor multiplicity in NDEA induced hepatic cancer bearing rats and this was evident from argyrophilic nucleolar organizer region (AgNORs) staining. Moreover, the uncontrolled cell proliferation was assessed by specific cell proliferative markers [proliferating cell nuclear antigen (PCNA) and Ki67] by immunofluorescence, immunoblot and analysis of mRNA expression. Simultaneously, we assessed the activity of TQ on G1/S phase cell cycle regulation with specific cell cycle proteins (p21WAF1/CIP1, CDK4, Cyclin D1 and Cyclin E) by immunoprecipitation in experimental rats. Treatment with TQ significantly reduced the detrimental alterations by abrogating cell proliferation, which strongly induced G1/S arrest in cell cycle transition. In conclusion, our results suggest that TQ has a potent anti proliferative activity by regulating the G1/S phase cell cycle transition and exhibit a beneficial role in the treatment of HCC.

HMGA2 Is a Driver of Tumor Metastasis

The non-histone chromatin-binding protein HMGA2 is expressed predominantly in the mesenchyme before its differentiation, but it is also expressed in tumors of epithelial origin. Ectopic expression of HMGA2 in epithelial cells induces epithelial-mesenchymal transition (EMT), which has been implicated in the acquisition of metastatic characters in tumor cells. However, little is known about in vivo modulation of HMGA2 and its effector functions in tumor metastasis. Here, we report that HMGA2 loss of function in a mouse model of cancer reduces tumor multiplicity. HMGA2-positive cells were identified at the invasive front of human and mouse tumors. In addition, in a mouse allograft model, HMGA2 overexpression converted nonmetastatic 4TO7 breast cancer cells to metastatic cells that homed specifically to liver. Interestingly, expression of HMGA2 enhanced TGFβ signaling by activating expression of the TGFβ type II receptor, which also localized to the invasive front of tumors. Together our results argued that HMGA2 plays a critical role in EMT by activating the TGFβ signaling pathway, thereby inducing invasion and metastasis of human epithelial cancers.

Abstract 4860: Chemopreventive effects of α-santalol on ultraviolet B induced skin tumorigenesis by modulating cell cycle regulators.

Abstract Previous studies from our laboratory have shown chemopreventive effects of 5% α-santalol, a sequiterpene isolated from the sandalwood oil in ultraviolet-B induced skin tumor multiplicity in SKH-1 hairless mice. However, this dosage did not have significant effect on tumor incidence. The objective of this study was to investigate the effects of α-santalol (10%, w/v in acetone) on UVB-induced skin tumor incidence in SKH-1 hairless mouse model and to identify the molecular targets in α-santalol mediated protection. Tumor initiation and promotion were carried out by UVB radiation (30 mJ/cm2/day for 5 days a week up to 30 week) that is in the range of human exposure to sunlight that can cause skin cancer. Topical treatment of mice with α-santalol causes a significant reduction in tumor multiplicity, tumor volume and tumor incidence, and also delayed tumor development. Histopathological analysis of hematoxylin-eosin stained skin sections showed that α-santalol pretreatment strongly inhibited UVB-induced epidermal hyperplasia and total thickness of the epidermis. The proliferation potential of skin and tumor tissue sections from UVB-induced carcinogenesis protocol was evaluated by immunohistochemical expression of proliferation marker PCNA. Both intensity of brown staining as well as the number of positively stained nuclei were reduced in skin tumors and adjacent skin tissues from α-santalol treated group when compared to control group. Western blot analysis of skin lysates showed that α-santalol decreases the level of different cyclin-dependent kinases and associated cyclins, Cdc25 phosphatases (Cdc25B and Cdc25C) together with an up-regulation of CDK inhibitor Cip1/p21. However, the level of Kip1/p27 and p53 did not show a significant difference. Our data suggested that α-santalol (10%, topical) is a safer and promising skin cancer chemopreventive agent through targeting different cell cycle regulators. (This work is supported by a Translational Cancer Center Research Grant, funded by the State of South Dakota). Citation Format: Sreevidya Santha, Chandradhar Dwivedi. Chemopreventive effects of α-santalol on ultraviolet B induced skin tumorigenesis by modulating cell cycle regulators. [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 4860. doi:10.1158/1538-7445.AM2013-4860

Abstract 3705: Chemopreventive and therapeutic activity of high anthocyanin-content blueberry against estrogen-mediated breast cancer.

Abstract Berries are gaining increasing importance lately for their chemopreventive and therapeutic potential against several cancers in pre-clinical studies. In our earlier studies, blueberry-supplemented diet has shown chemopreventive protection against 17β-estradiol-mediated mammary tumorigenesis. The 5 anthocyanidins in blueberry are presumable bioactives. In this study, we tested both preventive and therapeutic activities of high anthocyanin-content blueberry diet in ACI rat model against 17β-estradiol-mediated mammary tumorigenesis. The blueberry used was a blend of Tifblue and Rubel blueberry (50:50) (U.S. Highbush Blueberry Council, CA) which has substantially higher amount of anthocyanins than Berkley/Blue Jay/Bluecrop blueberries used in our earlier studies. To study the chemopreventive effect, female ACI rats (5-6-week old) were administered AIN-93M diet or diets supplemented with blueberry (2.5 and 5%, w/w) 2 weeks prior to 17β-estradiol implantation. To test for the therapeutic activity, when 5% of the animals in the control group developed palpable tumors, one half of the animals were provided diet supplemented with 5% blueberry while the other half were continued on control diet. Animals received diet and water ad libitum and palpated weekly to determine the effect of berry intervention on tumor latency and tumor growth. When the tumor incidence reached &amp;gt;90% in estrogen-treated group on control diet, all the animals were euthanized. Our data revealed i) the first tumor in control group appeared after 85 days, while this was delayed by 28 and 37 days in groups receiving 5% blueberry in chemopreventive and therapeutic mode, respectively. The effect was less pronounced (delay by 7 days) with 2.5% berry; ii) the tumor incidence was also decreased dose dependently (96% in control versus 85% and 61% with 2.5% and 5% blueberry in chemopreventive mode, 55% with 5% blueberry in therapeutic mode); iii) a 25% and 51% reduction in tumor multiplicity with 2.5% and 5% blueberry, respectively (57% reduction with 5% blueberry in therapeutic mode); and iv) 37% and 45% reduction in tumor volume with 5% blueberry in chemopreventive and therapeutic modes, respectively; the effect with 2.5% blueberry was only modest. These data suggest that the test blueberry is effective in inhibiting estrogen-mediated mammary tumorigenesis in both chemopreventive and therapeutic modes. In view of our recent findings of miRNA ‘signature’ in estrogen-induced mammary tumors and its favorable modulation by ellagic acid, we are analyzing modulation of miRNAs in the current study and its correlation with the tumor inhibition. (Supported by Highbush Blueberry Council, CA, Agnes Brown Duggan Fund and Helmsley Foundation). Citation Format: Jeyaprakash Jeyabalan, Farrukh Aqil, Radha Munagala, Ramesh Gupta. Chemopreventive and therapeutic activity of high anthocyanin-content blueberry against estrogen-mediated breast cancer. [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 3705. doi:10.1158/1538-7445.AM2013-3705

Abstract 176: Thymoquinone attenuates tumor development in a mouse model of Lynch syndrome.

Abstract Background: Hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) is caused by germline mutations of DNA mismatch repair proteins leading to microsatellite instability (MSI). Nigella sativa oil has known antitumorigenic effects and was identified in an in vitro screen for compounds that reduce microsatellite instability (C. Campregher, unpublished data). Here we used a mouse model for HNPCC (Villin-Cre Msh2loxP/loxP) to test the effect of thymoquinone (TQ), the active ingredient of Nigella sativa oil, on intestinal tumor development. Methods: 60 mice (20 per group) were treated for 10 months with chow containing TQ-low (37.5 mg/kg diet), TQ-high (375 mg/kg diet) or regular mouse chow. Serial tissue sections of paraffin embedded Swiss rolls of the entire intestine were H&amp;E stained and analyzed for tumor multiplicity, incidence and size. As tumor multiplicity in these mice is generally low (1-2 tumors/mouse) serial sections were prepared from the upper-, middle- and lower part of the paraffin block. Tumor size was scored as small, medium or large according to its presence on 1, 2 or all 3 sections of the block. DNA was isolated from microdissected normal intestinal and tumor tissue and tested for MSI by fragment analysis using a panel of six markers (Kabbarah 2003). Data were quantitated as nucleotide shift per marker (NSPM) and compared to germline DNA from mouse tails. Results: Treatment with TQ-low and TQ-high resulted in a significant reduction of tumor incidence from 94.4% to 57.9% (p&amp;lt;0.01) and 55.0% (p&amp;lt;0.006), respectively. TQ-low and TQ-high also reduced tumor multiplicity from 3.1 to 1.4 (p=0.003) and to 0.9 (p&amp;lt;0.001), respectively. The number of small and medium sized tumors was reduced in both treatment groups. The number of large tumors was not affected. Fragment analysis demonstrated global MSI in normal intestinal epithelial cells (on average 1.8 NSPM). TQ-low and TQ-high reduced the global MSI in normal intestinal epithelial cells to 0.9 and 0.9 NSPM, respectively. Tumor DNA displayed generally higher MSI than normal intestinal epithelial cells (2.7, 2.5, and 1.5 NSPM for control mice, TQ-low, and TQ-high, respectively) and was only reduced by TQ-high. Conclusion: In this mouse model of HNPCC TQ reduces the tumor incidence and tumor multiplicity by increasing replication fidelity in the Msh2-deficient intestinal epithelial cells. We propose TQ as a candidate compound for prevention of colorectal cancer in carriers of HNPCC mutations. Citation Format: Benedikt Kortüm, Christoph Campregher, Matthias Pinter, Michaela Lang, Rayko Evstatiev, Vineeta Khare, Melanie H. Kucherlapati, Winfried Edelmann, Christoph Gasche. Thymoquinone attenuates tumor development in a mouse model of Lynch syndrome. [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 176. doi:10.1158/1538-7445.AM2013-176

Abstract 2269: Metformin prolongs survival in a K-rasLA2 lung cancer mouse model by inhibiting cancer stem cells and tumor growth.

Abstract Mutations in K-Ras are characteristic of human lung cancers and occur almost exclusively in smokers. Metformin, which is widely prescribed for the treatment of type II diabetes mellitus, may reduce lung cancer incidence in diabetics. We previously showed that metformin prevents K-Ras-mediated lung tumorigenesis in mice exposed to tobacco-carcinogen in an AMPK-independent manner. To elucidate further mechanisms of tumor prevention and estimate the survival impact of metformin on mutant K-Ras-mediated-lung tumorigenesis, we treated mutant K-Ras transgenic mice (K-RasLA2), with oral metformin. Metformin reduced tumor multiplicity by 48.9% and increased overall survival by 34% (median survival 191 days (control) vs 257 days (metformin); HR=0.27; 95% CI 0.11-0.68; p=0.009). Metformin decreased the number of bronchioloalveolar cells (BASCs), which are recognized as the cells of origin for lung adenocarcinomas initiated by oncogenic K-ras, as well as expression of cancer stem cell markers such as OCT4 and SOX2. Markers of cellular proliferation such as cyclin D1 and Ki67 were also decreased in lung tumors from metformin-treated mice. Metformin inhibited activation of multiple receptor tyrosine kinases as well as Ras-Raf signaling. Collectively, these data confirm that metformin inhibits K-Ras-induced lung tumorigenesis and identifies tumor-initiating cells as targets of metformin whose inhibition may underlie the ability of metformin to prolong survival. Clinical chemoprevention trials using metformin are warranted for high-risk populations likely to develop K-Ras mutations such as current or former smokers. Citation Format: Hiroshi Kitagawa, Matthew Dallos, Brendan Quinn, Ajaikumar Kunnumakkara, Irem Dogan, Christine M. Hollander, Phillip A. Dennis. Metformin prolongs survival in a K-rasLA2 lung cancer mouse model by inhibiting cancer stem cells and tumor growth. [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 2269. doi:10.1158/1538-7445.AM2013-2269

MicroRNA-135b promotes cancer progression acting as a downstream effector of oncogenic pathways in colon cancer

BackgroundmicroRNAs (miRs) are small non-coding RNAs often deregulated in colorectal cancer. We aimed to identify miRs with a driver role in carcinogenesis altered by similar mechanisms in both human and mouse colorectal cancer. The goal was to use colorectal cancer mouse models for the preclinical development of anti-miRs as therapeutics. MethodsAzoximetane/dextran-sulphate (AOM/DSS) treated mice were used to study inflammation-associated colorectal cancer and CDX2P-NLS Cre;Apc+/loxP (CPC;Apc) mice were used to study sporadic APC-related colorectal cancer. Human inflammatory bowel disease associated (n=15) colorectal cancer and sporadic (n=62) colorectal cancer with their matched normal tissues were used for miR expression analysis. miR and gene expression profiling was assessed by nCounter technology. Anti-miR-135b probes for in-vivo studies were synthesised by Girindus. FindingsmiR profiling from AOM/DSS and CPC;Apc colorectal cancer showed that miR-135b is one of the most upregulated miR in both models. miR-135b was overexpressed in human inflammatory bowel disease and sporadic colorectal cancer and was associated with poor survival. Molecular studies in mouse embryo fibroblast and human colorectal cancer cell lines defined APC/β-catenin and SRC-PI3K as key pathways in miR-135b activation. miR-135b up-regulation resulted in reduced apoptosis and increased cell growth due to the downregulation of TGFRB2, DAPK1, APC, and FIH. miR-135b silencing in vivo reduced tumour multiplicity and tumour load in the AOM/DSS colorectal cancer model. Mice treated with anti-miR-135b showed well-differentiated tumours with an acinar pattern whereas tumours in the control groups showed poor differentiation and adenomatous pattern. Tumours in mice treated with anti-miR-135b showed reduced proliferation and increased apoptosis. InterpretationOur data suggest that miR-135b is a key molecule whose activation is downstream of driver genes frequently mutated in colorectal cancer. The in-vivo silencing of miR-135b shows preclinical efficacy with low toxicity and represents the first in-vivo study for use of anti-miRs in treatment of colorectal cancer. FundingUniversity of Glasgow.