Maria Marjorette O. Peña

Period 2 mutation accelerates ApcMin/+ tumorigenesis.

Colorectal cancer risk is increased in shift workers with presumed circadian disruption. Intestinal epithelial cell proliferation is gated throughout each day by the circadian clock. Period 2 (Per2) is a key circadian clock gene. Per2 mutant (Per2m/m) mice show an increase in lymphomas and deregulated expression of cyclin D and c-Myc genes that are key to proliferation control. We asked whether Per2 clock gene inactivation would accelerate intestinal and colonic tumorigenesis. The effects of PER2 on cell proliferation and β-catenin were studied in colon cancer cell lines by its down-regulation following RNA interference. The effects of Per2 inactivation in vivo on β-catenin and on intestinal and colonic polyp formation were studied in mice with Per2 mutation alone and in combination with an Apc mutation using polyp-prone ApcMin/+ mice. Down-regulation of PER2 in colon cell lines (HCT116 and SW480) increases β-catenin, cyclin D, and cell proliferation. Down-regulation of β-catenin along with Per2 blocks the increase in cyclin D and cell proliferation. Per2m/m mice develop colonic polyps and show an increase in small intestinal mucosa β-catenin and cyclin D protein levels compared with wild-type mice. ApcMin/+Per2m/m mice develop twice the number of small intestinal and colonic polyps, with more severe anemia and splenomegaly, compared with ApcMin/+ mice. These data suggest that Per2 gene product suppresses tumorigenesis in the small intestine and colon by down-regulation of β-catenin and β-catenin target genes, and this circadian core clock gene may represent a novel target for colorectal cancer prevention and control. (Mol Cancer Res 2008;6(11):1786–93)

A novel chemopreventive strategy based on therapeutic microRNAs produced in plants

Dear Editor, MicroRNAs (miRNAs) are small non-coding RNAs that play a critical role in regulation of gene expression in nearly all eukaryotic organisms, including mammals. In humans, an estimated 60% of all protein-coding genes are targeted by miRNAs, affecting virtually every physiological process in the body [1]. In addition, a diverse array of human diseases is associated with dysregulation of miRNAs [2]. In many forms of cancer, for example, certain miRNAs, termed tumor suppressor miRNAs, are downregulated in diseased cells. Restoration of the downregulated tumor suppressor miRNA has been shown to block one or more steps in oncogenesis in animal models and cell culture systems. Thus, the therapeutic potential of tumor suppressor miRNAs has been experimentally confirmed and is now widely recognized. However , systemic delivery of such therapeutic small RNAs in humans is challenging and numerous delivery options are currently under investigation. We have investigated the possibility of an effective oral delivery system for therapeutic miRNAs. It has long been known that ingested RNA from food sources is tak-en up by the digestive system in nematodes and insects and can control the expression of genes in those organisms [3]. More recent evidence suggests that a similar phenomenon might occur in humans and other mammals [4]. These data indicate that plant miRNAs from foods are absorbed by cells of the mammalian digestive tract and packaged into microvesicles, which protect them from degradation. The miRNAs are then trafficked via the bloodstream to a variety of tissues, where they are capable of regulating the expression of mammalian genes. Such work has generated considerable excitement because it raises the possibility of bioengineering edible plants to produce therapeutic miRNAs that could then be delivered to affected tissues by ingestion. However, the work has also generated controversy as several groups have subsequently reported being unable to detect ingest-ed plant miRNAs in mammalian tissues at levels significantly above background [5]. We addressed this controversy in experiments designed to both detect a therapeutic effect of ingested miRNAs and to demonstrate their uptake. Here we report that oral administration of a cocktail of tumor suppressor miRNAs reduced tumor burden in the well-established Apc Min/+ mouse model of colon cancer. The cocktail contains three validated tumor suppressor miRNAs (miR-34a, miR-143, and miR-145), synthesized with the exact nucleotide sequence of the mouse miRNAs, but with a methyl group on the 2′ position of the ribose of the 3′ terminal nucleotide, …

The Interaction of a High-Fat Diet and Regular Moderate Intensity Exercise on Intestinal Polyp Development in ApcMin/+ Mice

Diet and exercise are two environmental factors that can alter colon cancer risk. The purpose of this study was to determine if regular moderate-intensity treadmill exercise training could attenuate polyp formation in ApcMin/+ mice fed the Western-style diet. Four-week-old male ApcMin/+ mice (n = 12 per group) were assigned to AIN-76A Control, AIN-76A Exercise, Western Control, or Western Exercise treatment groups. Mice were weaned to these diets and either subjected to regular moderate-intensity treadmill exercise (18 m/min, 60 min/d, 6 d/wk) or remained sedentary for 6 weeks. Mice fed the Western-style diet consumed ∼14% more calories and had 42% more epididymal fat compared with mice fed the AIN-76A diet. Exercise had no effect on fat pad mass with either diet treatment. Exercise reduced total intestinal polyp number by 50% and the number of large polyps (>1 mm diameter) by 67% in AIN-76A–fed mice. The Western-style diet increased polyp number by 75% when compared with AIN-76A–fed mice, but exercise did not decrease polyp number or alter polyp size in mice fed the Western-style diet. Markers of systemic inflammation and immune system function were improved with exercise in mice fed the AIN-76A diet. Mice fed the Western-style diet showed more inflammation and immunosuppression, which were not completely ameliorated by exercise. These data suggest that the induction of adiposity, inflammation, and immunosuppression by the Western-style diet may compromise the beneficial effect of moderate-intensity exercise on the intestinal polyp burden in ApcMin/+ mice.

Linking tumor-associated macrophages, inflammation, and intestinal tumorigenesis: role of MCP-1

Tumor-associated macrophages are associated with poor prognosis in certain cancers. Monocyte chemoattractant protein 1 (MCP-1) is thought to be the most important chemokine for recruitment of macrophages to the tumor microenvironment. However, its role on tumorigenesis in a genetic mouse model of colon cancer has not been explored. We examined the role of MCP-1 on tumor-associated macrophages, inflammation, and intestinal tumorigenesis. Male Apc(Min/+), Apc(Min/+)/MCP-1(-/-) or wild-type mice were euthanized at 18 wk of age and intestines were analyzed for polyp burden, apoptosis, proliferation, β-catenin, macrophage number and phenotype, markers for cytotoxic T lymphocytes and regulatory T cells, and inflammatory mediators. MCP-1 deficiency decreased overall polyp number by 20% and specifically large polyp number by 45% (P < 0.05). This was consistent with an increase in apoptotic cells (P < 0.05), but there was no change detected in proliferation or β-catenin. MCP-1 deficiency decreased F4/80-positive cells in both the polyp tissue and surrounding intestinal tissue (P < 0.05) as well as expression of markers associated with M1 (IL-12 and IL-23) and M2 macrophages (IL-13, CD206, TGF-β, and CCL17) (P < 0.05). MCP-1 knockout was also associated with increased cytotoxic T lymphocytes and decreased regulatory T cells (P < 0.05). In addition, MCP-1(-/-) offset the increased mRNA expression of IL-1β and IL-6 in intestinal tissue and IL-1β and TNF-α in polyp tissue (P < 0.05), and prevented the decrease in SOCS1 expression (P < 0.05). We demonstrate that MCP-1 is an important mediator of tumor growth and immune regulation that may serve as an important biomarker and/or therapeutic target in colon cancer.

β-Catenin Induces β-TrCP-Mediated PER2 Degradation Altering Circadian Clock Gene Expression in Intestinal Mucosa of ApcMin/+ Mice

Proliferation of intestinal epithelial cells is rhythmic throughout the day. This temporal organization occurs through the interaction between the endogenous peripheral circadian clock and pathways controlling cell cycle progression. Per2, a core clock gene with tumour suppresser function, is critical to clock function and to the regulation of cellular proliferation. Circadian disruption, which increases colon cancer incidence, may do so by deregulating clock controlled epithelial cell proliferation. Increased expression of beta-catenin is a contributing cause of most familial and spontaneous human colon cancer and the cause of multiple intestinal neoplasia of the Apc(Min/+) mouse. Here we report that increased beta-catenin destabilizes PER2 clock protein by inducing beta-TrCP, an F-box protein of SCF ubiquitin E3 ligase. In the intestinal mucosa of the Apc(Min/)(+) mouse, the decrease in PER2 protein levels is associated with altered circadian rhythms of clock genes, Per1 and Per2, and clock controlled genes, Dbp and Wee1. These findings suggest that disruption of the peripheral intestinal circadian clock may be intimately involved in beta-catenin induced intestinal epithelial neoplastic transformation in both mouse and man.

Engineered resistance to camptothecin and antifolates by retroviral coexpression of tyrosyl DNA phosphodiesterase-I and thymidylate synthase

PurposeGene transfer of cDNA sequences that confer drug resistance can be used (1) to protect hematopoietic cells against the toxic effects of chemotherapy, (2) for in vivo enrichment of genetically engineered cells and (3) to protect cytotoxic T lymphocytes in drug-resistant immunotherapy approaches for the treatment of cancer. We have previously developed strategies to confer resistance to agents targeting thymidylate synthase (TS) and have now expanded our drug resistance strategies to include retroviral expression of tyrosyl-DNA phosphodiesterase (TDP-I), an enzyme recently implicated in the repair of topoisomerase-I (Top-I)/DNA lesions induced by camptothecin (CPT). The combination of TS and Top-I inhibition has been shown to be an effective treatment for several types of cancer.Materials and methodsRetroviral vectors were generated that individually encoded TS and TDP-I or that coexpressed both enzymes. Murine fibroblast and Chinese hamster lung transfectants were generated with the vectors and resistance to TS- and Top-I-directed inhibitors was tested. Murine bone marrow progenitor cells were also transduced using recombinant retroviruses encoding TS and TDP-I and the degree of drug resistance conferred to gene-modified cells was tested.ResultsEnforced expression of TDP-I increased TDP-I activity in gene-modified cells and conferred up to threefold resistance to CPT. The degree of resistance was dependent on the duration of drug treatment. Simultaneous expression of the TS gene encoding E. coli TS optimized for expression in mammalian cells (optecTS) and TDP-I conferred extremely high-level resistance to concurrent treatment with the TS-inhibitor BW1843U89 and CPT. Furthermore, by direct analysis of DNA fragmentation using the comet assay, substantial protection was conferred (fourfold) against DNA fragmentation associated with combination drug treatments by dual enzyme expression compared to non-modified cells. Hematopoietic progenitor assays of murine bone marrow cells transduced with retroviral vectors encoding TS and TDP-I showed that bone marrow cells could be protected from the cytotoxic effects of TS and Top-I inhibition.ConclusionsEnforced expression of optecTS and TDP-I conferred antifolate and CPT resistance to genetically modified cells. Additionally, this work further illustrated a role for TDP-I in the repair of dead-end Top-I complexes and implied that TDP-I expression analysis may aid in predicting the therapeutic effectiveness of the CPT class of compounds.

Developmental abnormalities in multiple proliferative tissues of ApcMin/+ mice

Germ‐line mutation of the Apc gene has been linked to familial adenomatous polyposis (FAP) that predisposes to colon cancer. ApcMin/+ mice, heterozygous for the Apc gene mutation, progressively develop small intestinal tumours in a manner that is analogous to that observed in the colon of patients with FAP ( Su et al. 1992; Fodde et al. 1994; Moser et al. 1995 ). We have studied the effects of Apc gene mutation on murine intestinal and extra‐intestinal, proliferatively active tissues. We have contrasted the histology to that of the age‐ and sex‐matched wild‐type C57BL/6 mice. Histological assessment of the normal appearing intestinal mucosa demonstrates minimal change in size of crypts. In contrast, villi are longer in the ileum of ApcMin/+ mice relative to C57BL/6 mice at 12 and 15 weeks of age. Vigorous splenic haematopoiesis in ApcMin/+ mice was seen at 12 and 15 weeks of age, as reflected by marked splenomegaly, increased splenic haematopoietic cells and megakaryocytes. Peripheral blood counts, however, did not differ between C57BL/6 and ApcMin/+ mice at 15 weeks of age. Lymphoid depletion in ApcMin/+ mice was characterized by diminished numbers of splenic lymphoid follicles and small intestinal Peyers patches. The ovaries of 12‐ and 15‐week‐old ApcMin/+ mice exhibited increased numbers of atretic follicles, and estrous cycling by serial vaginal smears showed tendency of elongation in the mutant mice during these age ranges. The testicles of 10‐week‐old ApcMin/+ mice showed increased numbers of underdeveloped seminiferous tubules. Collectively, these data suggest that, in addition to its obvious effects upon intestinal adenoma formation, Apc gene mutation causes impairment of developmental and apparent differentiation blockade in proliferative tissues, including those of the haematopoietic system, lymphoid and reproductive tract.

Effect of exercise on biological pathways in ApcMin/+ mouse intestinal polyps

Many epidemiological studies have demonstrated that level of exercise is associated with reduced colorectal cancer risk. Treadmill training can decrease Apc(Min/+) mouse intestinal polyp number and size, but the mechanisms remain unclear. Understanding the molecular changes in the tumor following exercise training may provide insight on the mechanism by which exercise decreases Apc(Min/+) mouse polyp formation and growth. The purpose of this study was to determine if exercise can modulate Apc(Min/+) mouse intestinal polyp cellular signaling related to tumor formation and growth. Male Apc(Min/+) mice were randomly assigned to control (n = 20) or exercise (n = 20) treatment groups. Exercised mice ran on a treadmill at a moderate intensity (18 m/min, 60 min, 6 days/wk, 5% grade) for 9 wk. Polyps from Apc(Min/+) mice were used to quantify markers of polyp inflammation, apoptosis, and beta-catenin signaling. Exercise decreased the number of macrophages in polyps by 35%. Related to apoptosis, exercise decreased the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells by 73% in all polyps. Bax protein expression in polyps was decreased 43% by exercise. beta-Catenin phosphorylation was elevated 3.3-fold in polyps from exercised mice. Moderate-intensity exercise training alters cellular pathways in Apc(Min/+) mouse polyps, and these changes may be related to the exercise-induced reduction in polyp formation and growth.

IL-33 promotes growth and liver metastasis of colorectal cancer in mice by remodeling the tumor microenvironment and inducing angiogenesis

Liver metastasis is the major cause of death from colorectal cancer (CRC). Understanding its mechanisms is necessary for timely diagnosis and development of effective therapies. Interleukin‐33 (IL‐33) is an IL‐1 cytokine family member that uniquely functions as a cytokine and nuclear factor. It is released by necrotic epithelial cells and activated innate immune cells, functioning as an alarmin or an early danger signal. Its role in invoking type 2 immune response has been established; however, it has contrasting roles in tumor development and metastasis. We identified IL‐33 as a potently upregulated cytokine in a highly metastatic murine CRC cell line and examined its role in tumor growth and metastasis to the liver. IL‐33 was transgenically expressed in murine and human adenocarcinoma and carcinoma cell lines and their growth and spontaneous metastasis to the liver were assessed in orthotopic models of CRC in wild‐type C57Bl/6 and Il33 knockout mice. The results showed that increased expression of IL‐33 in CRC cells enhanced their tumor take, growth, and liver metastasis. Tumor‐ rather than host‐derived IL‐33 induced the enhanced recruitment of CD11b+ GR1+ and CD11b+F4/80+ myeloid cells to remodel the tumor microenvironment by increased expression of mobilizing cytokines, and tumor angiogenesis by activating endothelial cells. IL‐33 expression was elevated in patient tumor tissues, induced early in adenoma development, and activated by pro‐inflammatory cytokines derived from the tumor microenvironment. The data suggest that tumor‐derived IL‐33 modulates the tumor microenvironment to potently promote colon carcinogenesis and liver metastasis, underscoring its potential as a therapeutic target.

Neonatal macrophages express elevated levels of interleukin‐27 that oppose immune responses

Microbial infections are a major cause of infant mortality worldwide because of impaired immune defences in this population. The nature of this work was to further understand the mechanistic limitations of the neonatal and infant immune response. Interleukin‐27 (IL‐27) is a heterodimeric cytokine of the IL‐12 family that is produced primarily by antigen‐presenting cells and is immunosuppressive toward a variety of immune cell types. We show that IL‐27 gene expression is elevated in cord blood‐derived macrophages relative to macrophages originating from healthy adults. We also evaluated the duration over which elevated IL‐27 gene expression may impact immune responses in mice. Age‐dependent analysis of IL‐27 gene expression indicated that levels of IL‐27 remained significantly elevated throughout infancy and then declined in adult mice. Flow cytometric analysis of intracellular cytokine‐stained splenocytes further confirmed these results. Interleukin‐27 may be induced during pregnancy to contribute to the immunosuppressive environment at the fetal–maternal interface because we demonstrate dose‐responsive gene expression to progesterone in macrophages. Neutralization of IL‐27 in neonatal macrophages improved the ability of these cells to limit bacterial replication. Moreover, neutralization of IL‐27 during incubation with the Mycobacterium bovis bacillus Calmette–Guérin vaccine augmented the level of interferon‐γ elicited from allogeneic CD4+ T lymphocytes. This suggests that blocking IL‐27 during vaccination and infection may improve immune responses in newborn and infant populations. Furthermore, mice will be a suitable model system to further address these possibilities.