Celestia Davis

Potent Modulation of Intestinal Tumorigenesis in Apcmin/+ Mice by the Polyamine Catabolic Enzyme Spermidine/Spermine N1-acetyltransferase

Intracellular polyamine pools are homeostatically maintained by processes involving biosynthesis, catabolism, and transport. Although most polyamine-based anticancer strategies target biosynthesis, we recently showed that activation of polyamine catabolism at the level of spermidine/spermine N(1)-acetyltransferase-1 (SSAT) suppresses tumor outgrowth in a mouse prostate cancer model. Herein, we examined the effects of differential SSAT expression on intestinal tumorigenesis in the Apc(Min/+) (MIN) mouse. When MIN mice were crossed with SSAT-overproducing transgenic mice, they developed 3- and 6-fold more adenomas in the small intestine and colon, respectively, than normal MIN mice. Despite accumulation of the SSAT product, N(1)-acetylspermidine, spermidine and spermine pools were only slightly decreased due to a huge compensatory increase in polyamine biosynthetic enzyme activities that gave rise to enhanced metabolic flux. When MIN mice were crossed with SSAT knock-out mice, they developed 75% fewer adenomas in the small intestine, suggesting that under basal conditions, SSAT contributes significantly to the MIN phenotype. Despite the loss in catabolic capability, tumor spermidine and spermine pools failed to increase significantly due to a compensatory decrease in biosynthetic enzyme activity giving rise to a reduced metabolic flux. Loss of heterozygosity at the Apc locus was observed in tumors from both SSAT-transgenic and -deficient MIN mice, indicating that loss of heterozygosity remained the predominant oncogenic mechanism. Based on these data, we propose a model in which SSAT expression alters flux through the polyamine pathway giving rise to metabolic events that promote tumorigenesis. The finding that deletion of SSAT reduces tumorigenesis suggests that small-molecule inhibition of the enzyme may represent a nontoxic prevention and/or treatment strategy for gastrointestinal cancers.

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, …

Response to 5-fluorouracil chemotherapy is modified by dietary folic acid deficiency in ApcMin/+ mice

5-Fluorouracil (5-FU) has been the foundation of advanced colorectal cancer treatment for over 40 years. The Apc(Min/+) mouse, which is genetically predisposed to intestinal neoplasia, was used to examine the effects of 5-FU in this system and the impact of dietary folic acid on those effects. 5-FU treatment resulted in a 60-80% reduction in tumor number. Clinically relevant toxicities, including myelosuppression and mucositis, are a part of this response. Tumor numbers rebounded completely following termination of 5-FU therapy, indicating that the drug inhibits tumor growth but does not eradicate them. In mice that were fed with a defined diet containing no folic acid (0 ppm), 5-FU not only induced regression of pre-existing tumors, but also inhibited tumor recovery following drug withdrawal. Our data indicate that a dietary folic acid deficiency, in promoting tumor regression and inhibiting tumor recovery, may enhance the therapeutic effects of 5-FU.

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.

Tissue-Specific shRNA Delivery: A Novel Approach for Gene Therapy in Cancer

A novel tissue-specific shRNA delivery system has been developed using cre-lox technology. Conditionally silenced pSico vector containing oligonucleotides of CD44shRNA and tissue-specific promoter-driven Cre-recombinase expression vector are packaged into transferrin-coated nanoparticles that can deliver shRNA into specific tumors. This system has strong potential in cancer therapy.

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.

Raltitrexed increases tumorigenesis as a single agent yet exhibits anti-tumor synergy with 5-fluorouracil in ApcMin/+ mice.

The thymidylate synthase (TS) inhibitors raltitrexed (RTX) and 5-fluorouracil (FUra) have shown promising anti-tumor activity in preclinical and clinical settings for the treatment of colorectal cancer. Though the effects of these two agents have been reasonably wellcharacterized in cell lines, knowledge of their modes of action in vivo is limited. Here, we utilize the ApcMin/+ mouse, an animal model of intestinal tumorigenesis, to study the effects of RTX treatment alone and in combination with FUra. Rather surprisingly, RTX monotherapy resulted in a dose dependent 4-10-fold increase in tumor number. The majority of these adenomas (74- 95%) were rather small (i.e., less than 1 mm in diameter) and exhibited loss of heterozygosity at the Apc locus, suggesting an increase in mutational events leading to tumor development. RTX augmented BrdU-labeling of crypt epithelial cells, and retarded the movement of these cells along the crypt-villus axis. Co-administration of FUra and RTX resulted in a significant reduction in tumor number compared to mice treated with either RTX or FUra alone (P < 0.0001). In addition, FUra abrogated the RTX-mediated increase in BrdU labeling. In all, the results show that RTX increases tumor burden in the ApcMin/+ mouse, yet enhances the anti-tumor effect of FUra. This is the first illustration of in vivo synergy of RTX and FUra in a genetically predisposed animal model. Possible mechanisms underlying the current observations are discussed.

Elevated interleukin-27 levels in human neonatal macrophages regulate indoleamine dioxygenase in a STAT-1 and STAT-3-dependent manner

Microbial infections are a major cause of infant mortality as a result of limitations in immune defences. Interleukin‐27 (IL‐27) is a heterodimeric cytokine produced primarily by leucocytes and is immunosuppressive toward lymphocytes and leucocytes. Our laboratory demonstrated that human neonatal macrophages express IL‐27 more abundantly than adult macrophages. Similarly in mice, IL‐27 expression is elevated early in life and maintained through infancy. To determine IL‐27‐regulated mechanisms that may limit immunity, we evaluated the expression of a number of genes in response to this cytokine in primary human neonatal macrophages. Indoleamine 2,3‐dioxygenase (IDO) gene expression was increased dose‐responsively by IL‐27. We have previously demonstrated inhibition of T‐cell proliferation and cytokine production by neonatal macrophage‐generated IL‐27, and IDO is often implicated in this negative regulation. An increase in IDO protein was demonstrated by immunofluorescence microscopy and was consistent with increased enzyme activity following treatment with IL‐27. Inclusion of a soluble receptor to neutralize endogenous IL‐27, decreased IDO expression and activity compared with untreated macrophages. In response to IL‐27, neonatal macrophages phosphorylate signal transdcuer and activator of transcription 1 (STAT‐1) and STAT‐3. Both transcription factors are recruited to the IDO regulatory region. STAT‐3 dominates during steady‐state regulation by lower levels of endogenous IL‐27 production. A shift to enhanced STAT‐1 recruitment occurs during increased levels of exogenously supplied IL‐27. These data suggest an interesting interplay of STAT‐1 and STAT‐3 to regulate IDO activity and immunosuppression in response to different levels of IL‐27 in the microenvironment of the immune response that may further our understanding of this interesting cytokine.

Hematopoietic Derived Cell Infiltration of the Intestinal Tumor Microenvironment in Apc Min /+ Mice

Tumors consist of a heterogeneous population of neoplastic cells infiltrated by an equally heterogeneous collection of nonneoplastic cells that comprise the tumor microenvironment. Tumor growth, invasion, and metastasis depend on multiple interactions between these cells. To assess their potential as therapeutic targets or vehicles for tumor specific delivery of therapeutic agents, we examined the contribution of bone marrow derived cells (BMDCs) to the intestinal tumor microenvironment. Hematopoietic stem cells expressing the enhanced green fluorescent protein (eGFP) were transplanted into lethally irradiated ApcMin/+ mice, and their engraftment was analyzed by confocal microscopy. The results showed abundant infiltration of eGFP cells into the small intestine, colon, and spleen compared to heart, muscle, liver, lung, and kidney. Within the intestine, there was a pronounced gradient of engraftment along the anterior to posterior axis, with enhanced infiltration into adenomas. Immunofluorescence analysis showed that osteopontin was expressed in tumor stromal cells but not in nontumor stromal populations, suggesting that gene expression in these cells is distinct. Tumor vasculature in ApcMin/+ mice was chaotic compared to normal intestinal regions. Our data suggest that BMDCs can be harnessed for tumor-targeted therapies to enhance antitumor efficacy.

Abstract C21: IL-33 promotes growth and liver metastasis of colorectal cancer in mice by remodeling the tumor microenvironment and inducing angiogenesis

The major cause of death from colorectal cancer (CRC) is liver metastasis and understanding its mechanisms is necessary for early diagnosis and development of new therapies. Interleukin-33 (IL-33) is an IL-1 cytokine that functions as a cytokine and nuclear factor. It is released by necrotic epithelial cells and activated innate immune cells, functioning as an alarmin or early danger signal. Its role in invoking type 2 immune response is firmly established; however, its role in tumor development and metastasis in only beginning to be elucidated. We identified IL-33 as a potently upregulated cytokine in highly metastatic CRC cells and examined its role in tumor growth and metastasis to the liver. We expressed IL-33 in murine and human CRC cell lines and their growth and spontaneous metastasis to the liver was assessed in orthotopic models of CRC in wild type and IL33 knockout mice. The results suggest that tumor- rather than host- derived IL-33 promoted growth and metastasis by inducing expression of cytokines that stimulated mobilization of CD11b+ GR1+ and CD11b+F4/80+ myeloid cells to remodel the tumor microenvironment, and increase tumor angiogenesis, potentially by activating endothelial cells. Conversely, pro-tumorigenic cytokines derived from the microenvironment induced IL-33 expression in tumor cells to create a sustained pro-tumorigenic loop that promotes tumor growth and metastasis. Our findings underscore the potential of IL-33 as a novel and viable therapeutic target for CRC and liver metastasis. Citation Format: Maria Marjorette O. Pena, Yu Zhang, Celestia Davis, James C. Ryan, Diego Altomare, Sapana Shah, Daniel Titus Hughes. IL-33 promotes growth and liver metastasis of colorectal cancer in mice by remodeling the tumor microenvironment and inducing angiogenesis. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C21.