Jennifer K.L. Colby

Paracrine Overexpression of Insulin-Like Growth Factor-1 Enhances Mammary Tumorigenesis in Vivo

Insulin-like growth factor-1 (IGF-1) stimulates proliferation, regulates tissue development, protects against apoptosis, and promotes the malignant phenotype in the breast and other organs. Some epidemiological studies have linked high circulating levels of IGF-1 with an increased risk of breast cancer. To study the role of IGF-1 in mammary tumorigenesis in vivo, we used transgenic mice in which overexpression of IGF-1 is under the control of the bovine keratin 5 (BK5) promoter and is directed to either the myoepithelial or basal cells in a variety of organs, including the mammary gland. This model closely recapitulates the paracrine exposure of breast epithelium to stromal IGF-1 seen in women. Histologically, mammary glands from transgenic mice were hyperplastic and highly vascularized. Mammary glands from prepubertal transgenic mice had significantly increased ductal proliferation compared with wild-type tissues, although this difference was not maintained after puberty. Transgenic mice also had increased susceptibility to mammary carcinogenesis, and 74% of the BK5.IGF-1 mice treated with 7,12-dimethylbenz[a]anthracene (20 microg/day) developed mammary tumors compared with 29% of the wild-type mice. Interestingly, 31% of the vehicle-treated BK5.IGF-1 animals, but none of the wild-type animals, spontaneously developed mammary cancer. The mammary tumors were moderately differentiated adenocarcinomas that expressed functional, nuclear estrogen receptor at both the protein and mRNA levels. These data support the hypothesis that tissue overexpression of IGF-1 stimulates mammary tumorigenesis.

Genetic Reduction of Insulin-like Growth Factor-1 Mimics the Anticancer Effects of Calorie Restriction on Cyclooxygenase-2–Driven Pancreatic Neoplasia

Risk of pancreatic cancer, the fourth deadliest cancer in the United States, is increased by obesity. Calorie restriction (CR) prevents obesity, suppresses carcinogenesis in many models, and reduces serum levels of IGF-1. In the present study, we examined the impact of CR on a model of inflammation-associated pancreatitis and pancreatic dysplasia, with a focus on the mechanistic contribution of systemic IGF-1. Administration of a 30% CR diet for 14 weeks decreased serum IGF-1 levels and hindered pancreatic ductal lesion formation and dysplastic severity, relative to a higher calorie control diet, in transgenic mice overexpressing COX-2 [bovine keratin-5 promoter (BK5.COX-2)]. These findings in CR mice correlated with reductions in Ki-67–positive cells, vascular luminal size, VEGF expression, and phosphorylation and total expression of downstream mediators of the IGF-1 pathway. Cell lines derived from BK5.COX-2 ductal lesions (JC101 cells) formed pancreatic tumors in wild-type FVB mice that were significantly reduced in size by a 14-week CR regimen, relative to the control diet. To further understand the impact of circulating levels of IGF-1 on tumor growth in this model, we orthotopically injected JC101 cells into liver-specific IGF-1–deficient (LID) mice. The approximate 65% reduction of serum IGF-1 levels in LID mice resulted in significantly decreased burden of JC101 tumors, despite modestly elevated levels of circulating insulin and leptin. These data show that CR prevents development of dysplasia and growth of pancreatic cancer through alterations in IGF-1, suggesting that modulation of this pathway with dietary and/or pharmacologic interventions is a promising pancreatic cancer prevention strategy. Cancer Prev Res; 4(7); 1030–40. ©2011 AACR.

Overexpression of cyclooxygenase-2 (COX-2) in the mouse urinary bladder induces the expression of immune and cell proliferation related genes

The mechanisms whereby cyclooxygenase‐2 (COX‐2) overexpression may contribute to bladder carcinogenesis remain unknown. We recently developed a transgenic mouse model overexpressing COX‐2 under the control of a bovine keratin 5 (BK5) promoter causing a high incidence of transitional cell hyperplasia (TCH) in the bladder with a proportion of lesions progressing to invasive carcinoma. Microarray gene analysis was employed to determine the effects of COX‐2 overexpression on gene expression profiles in the urinary bladder. Statistical analysis revealed that 70 genes were upregulated and 60 were downregulated by twofold or more in bladders from transgenic compared to wild‐type mice. Expression Analysis Systematic Explorer (EASE) analysis revealed that genes associated with Immune/Stress Response and Cell Cycle/Proliferation biological processes were overexpressed in the transgenic mice. Relevant downregulated genes included three transforming growth factor (TGF)‐β related genes, Tgfb2, Tgfb3, and Tgfbi. The growth factor epiregulin was the most highly induced gene among those validated by qRT‐PCR in TCH of BK5.COX‐2 mouse bladders in parallel with increased staining for Ki67. Prostaglandin E2 (PGE2) directly induced the expression of epiregulin mRNA in bladders from wild‐type FVB mice ex vivo. We further determined that recombinant epiregulin increased both cell proliferation and Erk phosphorylation in UMUC‐3 bladder cancer cells. These results indicate that the response of the mouse urinary bladder to elevated COX‐2 expression includes enhanced inflammatory response and induction of cell proliferation. The growth factor epiregulin may play a role in bladder carcinogenesis and may serve as a novel target for the prevention and treatment of bladder cancer.

P53 genotype as a determinant of ER expression and tamoxifen response in the MMTV-Wnt-1 model of mammary carcinogenesis

Clinical studies show that estrogen receptor-α (ER) expressing tumors tend to have better prognosis, respond to antiestrogen therapy and have wild-type p53. Conversely, tumors with inactivating mutations in p53 tend to have worse outcomes and to be ER-negative and unresponsive to antihormone treatment. Previous studies from our laboratory have shown that p53 regulates ER expression transcriptionally, by binding the ER promoter and forming a complex with CARM1, CBP, c-Jun, RNA polymerase II and Sp1. In this study, the MMTV-Wnt-1 transgenic mouse model was used to demonstrate that p53 regulation of ER expression and function is not solely an in vitro phenomenon, but it is also operational in mammary tumorigenesis in vivo. The expression of ER and the ability to respond to tamoxifen were determined in mammary tumors arising in p53 wild type (WT) or p53 heterozygous (HT) animals carrying the Wnt-1 transgene. In p53 WT mice, development of ER-positive tumors was delayed by tamoxifen treatment, while tumors arising in p53 HT mice had significantly reduced levels of ER and were not affected by tamoxifen. P53 null tumors were also found in the p53 HT mice and these tumors were ER-negative. ER expression was upregulated in mouse mammary tumor cell lines following transfection with WT p53 or treatment with doxorubicin. These data demonstrate that p53 regulates ER expression in vivo, and affects response to tamoxifen. Results also provide an explanation for the concordant relationship between these prognostic proteins in human breast tumors.

ALOX15 as a suppressor of inflammation and cancer: Lost in the link.

Mounting evidence supports a mechanistic link between inflammation and cancer, especially colon cancer. ALOX15 (15-lipoxygenase-1) plays an important role in the formation of key lipid mediators (e.g., lipoxins and resolvins) to terminate inflammation. ALOX15 expression is downregulated in colorectal cancer (CRC). Intestinally-targeted transgenic expression of ALOX15 in mice inhibited dextran sodium sulfate-induced colitis from promoting azoxymethane- induced colorectal tumorigenesis, demonstrating that ALOX15 can suppress inflammation-driven promotion of carcinogen-induced colorectal tumorigenesis and therefore ALOX15 downregulation during tumorigenesis is likely to enhance the link between colitis and colorectal tumorigenesis. ALOX15 suppressed the TNF-α, IL-1β/NF-κB, and IL-6/STAT3 signaling pathways, which play major roles in promotion of colorectal cancer by chronic inflammation. Defining ALOX15s regulatory role in colitis-associated colorectal cancer could identify important molecular regulatory events that could be targeted to suppress promotion of tumorigenesis by chronic inflammation.

The Role of PPAR-δ in Metabolism, Inflammation, and Cancer: Many Characters of a Critical Transcription Factor

Peroxisome proliferator-activated receptor-delta (PPAR-δ), one of three members of the PPAR group in the nuclear receptor superfamily, is a ligand-activated transcription factor. PPAR-δ regulates important cellular metabolic functions that contribute to maintaining energy balance. PPAR-δ is especially important in regulating fatty acid uptake, transport, and β-oxidation as well as insulin secretion and sensitivity. These salutary PPAR-δ functions in normal cells are thought to protect against metabolic-syndrome-related diseases, such as obesity, dyslipidemia, insulin resistance/type 2 diabetes, hepatosteatosis, and atherosclerosis. Given the high clinical burden these diseases pose, highly selective synthetic activating ligands of PPAR-δ were developed as potential preventive/therapeutic agents. Some of these compounds showed some efficacy in clinical trials focused on metabolic-syndrome-related conditions. However, the clinical development of PPAR-δ agonists was halted because various lines of evidence demonstrated that cancer cells upregulated PPAR-δ expression/activity as a defense mechanism against nutritional deprivation and energy stresses, improving their survival and promoting cancer progression. This review discusses the complex relationship between PPAR-δ in health and disease and highlights our current knowledge regarding the different roles that PPAR-δ plays in metabolism, inflammation, and cancer.

Oxygenated lipid signaling in tumor-associated macrophages—focus on colon cancer

Polyunsaturated fatty acids (PUFAs) are enzymatically converted to a variety of bioactive products through insertion of molecular oxygen. PUFA-derived mediators can have either inflammatory or anti-inflammatory/pro-resolving properties, depending upon their specific structures. The relative harm or benefit of these mediators can also be tissue and context dependent. These mediators play important roles in maintaining homeostasis and their dysregulation is involved in pathogenesis of cancers, especially those associated with chronic inflammation. There is a well-established link between colorectal cancer (CRC) and chronic inflammation. The colon harbors a large population of immune cells, which must be tightly regulated in order to maintain the balance between pathogenic and commensal microbes in the gut. Macrophages are key to the process of distinguishing between potentially harmful antigens/microbes and benign or beneficial signals. Macrophages are often associated with tumors (tumor-associated macrophages (TAMs), including CRC. There is some debate as to the prognostic significance of these TAMs in CRC, with some work suggesting a beneficial impact. The purpose of this review is to give an overview of what is currently known regarding PUFA-derived mediator signaling in tumor-associated macrophages in CRC.

Abstract 2224: Increased 15-lipoxygenase-1 activity limits tumor development in the azoxymethane mouse model of colon cancer: impact of omega-3-acid ethyl esters

Pro-inflammatory signaling has been shown to promote colorectal tumorigenesis and is a target for the development of effective chemopreventive approaches. The specialized pro-resolving lipid mediators (SPMs, e.g. resolvins), bioactive metabolites of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), actively terminate inflammation and have been proposed to possibly contribute to the anti-tumorigenic effects of DHA and EPA. The enzyme 15-lipoxygenase-1 (ALOX15) is a key biosynthetic enzyme in generation of resolvins. However, 15-LOX-1 expression is commonly lost during human CRC tumorigenesis starting in premalignant stages via transcriptional mechanisms independent of substrate availability. The impact of ALOX15 on DHA and EPA’s effects on tumorigenesis remain unknown. Mice with intestinal epithelium-specific expression of human ALOX15 (15-LOX-1-gut mice) and wild-type FVB controls were injected with azoxymethane (AOM, 7.5mg/kg) once weekly for 6w and followed for 20w. Mice were fed diet with 1% omega-3-acid ethyl esters (O3AEE, a pharmaceutical grade fish oil preparation of EPA and DHA ethyl esters) or control diet starting 3w before initiation with AOM. Colonic tumors developed in 10 of the 13 (77%) wild type (WT) mice fed control diet, 5 of the 10 (50%) WT mice fed O3AEE diet, 5 of the 12 (42%) 15-LOX-1-gut mice fed control diet, and 3 of the 10 (30%) 15-LOX-1-gut mice fed control diet. Lipid mediator levels were measured by liquid chromatography/ tandem mass spectrometry (LC-MS/MS). The SPMs resolvin E1 and D2 (RvE1, RvD2) as well as the pathway intermediates 18-HEPE and 17-HDHE were increased in ALOX15-gut mice on O3AEE (see Table; data are shown as ng/mg protein; mean ± SEM). Our results demonstrate that ALOX15 activity is important to DHA and EPA formation of resolvins and inhibition of colonic tumorigenesis. Supported by grants CPRIT RP150195 and NIH RO1 R01CA195686 Citation Format: Xiangsheng Zuo, Jennifer K. Colby, Fuyao Liu, Shen Gao, Ling Wu, Jonathan C. Jaoude, Micheline J. Moussalli, Lin Tan, Peiying Yang, Imad Shureiqi. Increased 15-lipoxygenase-1 activity limits tumor development in the azoxymethane mouse model of colon cancer: impact of omega-3-acid ethyl esters [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 2224. doi:10.1158/1538-7445.AM2017-2224

Abstract 2883: Genetic reduction of circulating IGF-1 inhibits growth of COX-2- and Kras-induced pancreatic tumors in orthotopic transplant models

Chronic pancreatitis dramatically increases the risk of pancreatic cancer, the 4 th deadliest malignancy in the US. KRAS mutations, the predominant genetic lesion found in pancreatic cancer, are detectable in 30% of patients with chronic pancreatitis. We previously found that calorie restriction (CR) protected against the tumorigenic effects of chronic pancreatitis in the cyclooxygenase (COX)-2-driven (BK5.COX-2) transgenic mouse model of pancreatic cancer. The protection conferred by CR was associated with dramatically reduced circulating levels of insulin-like growth factor (IGF)-1, an established mitogen in a variety of cancer cell lines. We hypothesized that circulating IGF-1 levels have a crucial role in tumor progression in both pancreatitis- and KRAS-induced pancreatic cancer. To test this, mouse pancreatic cancer cells derived from either a BK5.COX-2 transgenic mouse (JC101) or a Kras G12D /INK4a +/− mouse (NB508) were orthotopically transplanted in liver-specific IGF-1-deficient (LID, n=16 per cell line) and wild-type (WT, n=11 per cell line) mice. LID mice were randomized to receive Alzet miniature osmotic pumps (implanted subcutaneously) continuously infusing either vehicle (n=8) or 1 µg/hr of recombinant human IGF-1 (rhIGF-1, Increlex®, Tercica, Inc., Brisbane, CA; n=8). WT mice were infused with vehicle. Mice were euthanized 28 days after tumor and pump implantation, and blood was collected and tumors were weighed and fixed in 10% normal buffered formalin. Immunohistochemical staining for Ki-67 was performed on paraffin-embedded tumors to assess proliferation. In LID mice serum IGF-1 levels were reduced approximately 70% relative to WT (p 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 2883.

Abstract A102: Genetic reduction of circulating IGF‐1 decreases growth of pancreatic tumors in an orthotopic transplant model

Pancreatic cancer is the 4th deadliest cancer in the U.S. Obesity has emerged as a significant and modifiable risk factor for pancreatic cancer. Calorie restriction (CR) is an anti‐obesity intervention that has been shown in many model systems to have potent anticancer effects. Levels of bioavailable insulin‐like growth factor (IGF)‐1, an established mitogen in a variety of cancer cell lines, increase in response to positive energy balance and decrease with CR. We hypothesized that the genetic reduction of circulating IGF‐1 levels seen in liver‐specific IGF‐1‐deficient (LID) mice would diminish the tumor burden of orthotopically transplanted pancreatic cancer cells, similar to CR, while administration of exogenous IGF‐1 would restore the tumor burden. To test this hypothesis, mouse pancreatic cancer cells (JC101, derived from our BK5‐COX‐2 transgenic mouse model of spontaneous pancreatic cancer) were orthotopically transplanted in LID and littermate control (LC) mice. LID mice were also randomized to receive Alzet miniature osmotic pumps (implanted subcutaneously) continuously infusing either vehicle (n=8) or 1 µg/hr of recombinant human IGF‐1 (Increlex®, Tercica, Inc., Brisbane, CA; n=8). LC mice were infused with vehicle (n=8). Mice were killed 28 days after injection, blood was collected by cardiac puncture and serum samples were stored at −80°C, and tumors were weighed and fixed in 10% normal buffered formalin for histological and immunohistochemical (IHC) analysis. IHC staining was performed on paraffin‐embedded tissues using antibodies against Ki67 and cyclin D1 to assess proliferation. LID mice exhibited significantly smaller tumors (0.20 ± 0.02 g) than LC mice (0.45 ± 0.11 g, p Citation Information: Cancer Prev Res 2010;3(1 Suppl):A102.