Molecular events involved in the increased expression of matrix metalloproteinase-9 by T lymphocytes of mammary tumor-bearing mice

Matrix metalloproteinases (MMPs) are a family of extracellular proteinases whose contributions to cancer progression have been studied because of their matrix-degrading abilities and elevated expression in advanced stage tumors. Recent findings suggest a role for MMPs during the multiple stages of tumor progression including establishment and growth, migration, invasion, metastasis, and angiogenesis. MMP-9 regulation at the molecular level can be studied by measuring the effect(s) of a variety of physiological and pharmacological agents on cells. Multiple signaling molecules such as protein kinase C, pertussis toxin-sensitive guanine nucleotide-binding protein G, and protein tyrosine kinases are known to mediate the secretion of MMPs in cell lines. We previously reported an upregulation of MMP-9 in T cells of mammary tumor-bearing mice. In this study, pharmacologic inhibitors were used to dissect the signaling pathways involved in the upregulation of MMP-9 in the splenic T cells of normal and mammary tumor-bearing mice. Staurosporine, a protein kinase inhibitor, stimulated MMP-9 secretion by normal T lymphocytes, while the constitutively high levels of MMP-9 produced by tumor bearers' T cells were decreased by Genistein, a specific tyrosine kinase inhibitor, and Rottlerin, a PKC inhibitor. Using a NF-kappaB specific probe to the murine MMP-9 promoter, electromobility shift assays of nuclear proteins from normal and tumor bearers' splenic T cells revealed a pattern of higher intensity bands from the tumor bearers' nuclear extracts, indicating a greater amount of these transcription factors bound to the recognition motif. When mammary tumor bearers' T cells were cultured with the NF-kappaB inhibitors, N-p-Tosyl-L-lysine chloromethyl ketone hydrochloride and Bay 11-7082, there was a subsequent decreased production of MMP-9. These results suggest that the tumor burden may be activating various signaling pathways within splenic T lymphocytes to upregulate MMP-9 expression

Role of the proteasome in the downregulation of transcription factors NFkappaB and C/EBP in macrophages from tumor hosts

Macrophages from mice bearing advanced mammary tumors are critically impaired in their immune functions, exhibiting reduced expression at the mRNA and protein levels of the crucial transcription factors, nuclear factor kappaB (NFkappaB) and CCAAT enhancer binding protein (C/EBP). We have previously shown that tumor-derived factors such as transforming growth factor beta (TGFbeta) and prostaglandin E2 (PGE2) modulate NFkappaB and C/EBP expression in macrophages. Transcriptional, post-transcriptional, translational and/or post-translational mechanisms may also play a role in altered levels of NFkappaB and C/EBP in macrophages from tumor hosts, contributing to impaired inflammatory response. One of the post-translational mechanisms that may tune down or recycle proteins in cells is the proteasomal pathway. Since upregulation of ubiquitin/proteasomal pathways has been described under cancer-induced cachexia, we examined the possible role of this proteolytic machinery in the decrease of NFkappaB and C/EBP proteins in macrophages from tumor hosts. Using MG-132 proteasome inhibitor to block the proteasome machinery in macrophages from normal and tumor-bearing animals we found that macrophages from tumor hosts display higher ubiquitination and proteolysis compared to those from normal mice and also that NFkappaB and C/EBP downregulation is reversed in these treated cells. Thus, proteasome degradation may contribute, at least in part, to NFkappaB and C/EBP impairment in macrophages from tumor-bearers

IL-11-induced reduction of C/EBP transcription factor binding may contribute to the IL-12 downregulation in tumor-bearing mice

Using a mammary tumor model syngeneic to BALB/c mice, we have characterized several tumor-derived factors. We now report that the DA-3 cell line derived from this tumor, as well as the in vivo tumor itself, express IL-11. The expression of IL-11 in the tumor is detectable at the transcriptional and translational levels, as evidenced by RT-PCR and Western blots. Using a murine IL-11 ELISA, we observed no differences in IL-11 production between normal and tumor-bearer's macrophages or T cells, with or without activation. Interestingly, elevated levels of IL-11 were found in the sera of tumor-bearers, when compared to normal animals and even higher levels of IL-11 were detected in the tumor cystic fluid. Macrophages from mice bearing large mammary tumors show an impaired production of IL-12 and NO, whereas T cells from the same animals display a deficient production of IFN-gamma. Pretreatment of normal macrophages with IL-11 resulted in no decrease in NO production, nor an impaired production of IFN-gamma was observed in normal T cells upon pretreatment with IL-11. However, pretreatment of normal macrophages with IL-11 resulted in a decreased production of IL-12, as revealed by ELISA and RT-PCR. Electromobility shift assays showed decreased binding of the transcription factor C/EBP to the IL-12p40 promoter of LPS-activated macrophages from normal animals, upon pretreatment with IL-11. In contrast, no differences were observed in the levels of NFkappaB binding under the same experimental conditions. Our results suggest that tumor-derived IL-11 may play a role in the depressed IL-12 production by macrophages, leading to the impaired immune functions observed during mammary tumorigenesis

GM-CSF up-regulates the expression of CCL2 by T lymphocytes in mammary tumor-bearing mice

CP-1/CCL2 (monocyte chemoattractant protein-1/CC chemokine ligand 2) is a β or CC chemokine that is expressed by a variety of cell types, including fibroblasts, endothelial, smooth muscle, and glial cells. In addition, cells involved in immunity, such as monocytes/macrophages, neutrophils, and eosinophils have also been shown to express this chemoattractant. Using a murine model of the D1-DMBA-3 mammary adenocarcinoma, we demonstrated the unique production of CCL2 by splenic T lymphocytes from tumor-bearing animals. Because this tumor produces GM-CSF, and this factor is also up-regulated in the B lymphocytes of tumor-bearing mice, we looked at the ability of GM-CSF to induce CCL2 production by T cells. Treatment of normal and tumor bearers' T cells with GM-CSF resulted in an increased secretion of this chemokine. This up-regulation was seen with or without stimulation by Concanavalin A, although these treatments were additive in their effects. The induction of CCL2 was studied at the molecular level by analyzing the effect(s) of a variety of physiological and pharmacological agents on cultured T cells. These results suggest that the tumor-derived factor GM-CSF activates various signaling pathways within splenic T cells to up-regulate CCL2 expression

Diminished PKC activity and decreased binding of transcription factors are involved in the impaired production of nitric oxide by macrophages from tumor-bearing mice

In previous studies we have shown that peritoneal macrophages (PEM) from mammary tumor-bearing BALB/c mice (T-PEM) display a diminished ability to lyse tumor cells upon stimulation with LPS, a phenomenon that is associated to a lower production of nitric oxide, and that is reverted upon costimulation with IFN-gamma. The reduced lytic activity and NO production displayed by T-PEM upon LPS activation were earlier shown by us to be due to a diminished transcription of the inducible nitric oxide synthase (iNOS) gene. In the present study, we have investigated the participation of possible signaling molecules and transcription factors - PKC, NF-kappaB, C/EBP and IRF-1 - in the downregulation of NO production in LPS-activated T-PEM. It was found that PKC activity was greatly reduced in T-PEM as compared to normal macrophages, and did not respond to activation. Interestingly, the different PKC isozyme levels were not significantly altered in T-PEM, with the exception of PKC delta. Alterations in the binding activity of the transcription factors NF-kappaB and C/EBP appeared to be involved in the reduced transcription of iNOS previously observed in T-PEM after LPS activation. These results provide evidence that reductions in iNOS transcription secondary to alterations in cell signaling may be responsible for the diminished capacity of macrophages of LPS-activated tumor-bearers to produce NO and lyse tumor targets

Abstract 792: Tumor-induced thymic atrophy: Alteration in interferons and Jak/Stats signaling pathways

Abstract The thymus is the major site of T cell differentiation and a key organ of the immune system. Most important, it provides a specialized environment for the selection of rearranged clones that will function appropriately in the adaptive immune response. Thymic involution has been observed in several model systems; including graft-vs-host disease, aging, and tumor development, however, the mechanisms involved in this phenomenon remain to be elucidated. Previous results from our laboratory have reported that the severe thymic atrophy and impaired T cell development seen in mammary tumor bearers are associated with an arrest in at least two steps of T cell differentiation, changes in the levels of crucial cytokines expressed in the thymus microenvironment, and a progressive increase in apoptosis during the tumor development mainly due to downregulation of important molecules that control programmed cell death. Cytokines regulate numerous aspects of hematopoiesis via activation of the Jak/Stat pathways. In the present study we have used our mammary tumor model to investigate whether changes in the levels of cytokines in the thymus could affect the normal expression of the aforementioned pathways. RNA and protein analysis revealed an overexpression of the different interferons, a downregulation of most of the Jak/Stat pathways, and an increased expression of several suppressors of cytokine signaling (SOCS) in the thymuses of tumor bearers. Collectively, our data suggest a mechanism by which the impaired Jak/Stat signaling pathways observed in the whole thymus of tumor-bearing mice could be contributing to the abnormal T cell development and apoptosis observed during the tumor-induced thymic atrophy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 792. doi:10.1158/1538-7445.AM2011-792</jats:p

Tumor microenvironment profoundly modifies functional status of macrophages: Peritoneal and tumor-associated macrophages are two very different subpopulations

Macrophages are key players in the inflammatory response. In this study, we tested the hypothesis that although all macrophage subpopulations in tumor hosts are affected by the disease, it is the close proximity to the tumor that induces major alterations in these cells. We compared tumor-associated macrophages (TAMs) with peritoneal macrophages from mice bearing D1-DMBA-3 mammary tumors (T-PEMs). Our results show that TAMs downregulate IL-12p70 but upregulate IL-12p40, IL-23, IL-6 and IL-10. Some NFκB and C/EBP transcription factors family members are decreased in TAMs; however NFκBp50 homodimers, STAT1/pSTAT1 and STAT3/pSTAT3 are overexpressed. Furthermore, while TAMs block T-cell proliferation and are more prone to apoptosis compared to T-PEMs, both types of macrophages have an impaired phagocytic capacity. Moreover, TAMs constitutively express iNOS and produce nitric oxide but do not express arginase and are Gr-1(high) and CD11b(low). Collectively, our analysis of two spatially distinct macrophage subpopulations in tumor-bearing mice revealed that the tumor modulates them differently into two molecularly and functionally dissimilar macrophage subpopulations

Induction of proinflammatory mediators by CHI3L1 is reduced by chitin treatment: decreased tumor metastasis in a breast cancer model

Disseminated metastasis accounts for over 90% of breast cancer deaths. Recently, elevated serum levels of a glycoprotein known as chitinase-3 like-protein-1 (CHI3L1) has been correlated with poor prognosis and shorter survival of patients with metastatic breast cancer. In this study, we show that there are increased levels of CHI3L1 in plasma of tumor-bearing mice and that both tumor cells and immune cells express and secrete CHI3L1. However, the biological and physiological functions of CHI3L1 are still unclear. We demonstrate that while CHI3L1 has an inhibitory role in the expression of interferon-gamma (IFN-γ), CHI3L1 up-regulates pro-inflammatory mediators, C-chemokine ligand 2 (CCL2), Chemokine CX motif ligand 2 (CXCL2) and matrix metalloproteinase-9 (MMP-9) all of which contribute to tumor growth and metastasis. We found that in vitro inhibition of CHI3L1 by siRNA suppressed the production of CCL2, CXCL2 and MMP-9 by macrophages. In vivo treatment of mammary tumor-bearing mice with chitin (β-(1–4)-poly-N-acetyl D-glucosamine), a TH 1 adjuvant and a ligand for CHI3L1, promoted immune effector functions with increased production of IFN-γ and decreased CCL2, CXCL2 and MMP-9 expression. In vivo administration of chitin to mammary tumor-bearing mice significantly decreased lung metastasis. These studies show that CHI3L1 plays a role in tumor progression and that chitin can inhibit the pleiotropic effects of CHI3L1 giving support to the idea that CHI3L1 is a useful therapeutic target for treatment of breast cancer

Abstract 3496: Breast cancer and obesity impact the lipid composition of breast adipose tissue: a preliminary study using shotgun lipidomics

Abstract Obesity, an established risk factor for breast and other cancers, is associated with systemic inflammation and increased visceral adipose tissue. Adipose tissue is a normal constituent of the breast; however, the role of breast adipose tissue in breast cancer development, especially in the context of obesity, has not been addressed before. There is no information on the lipid composition of different fat depots in the body, especially in the context of obesity, and even less among obese tumor hosts. The study of the lipid composition of breast adipose tissue in diet-induced obese (DIO) tumor-bearing and normal mice and its impact in breast cancer progression is novel and has not been previously examined. New profiling methods employing shotgun lipidomics, a technique employed in mass spectrometric analysis using the direct loading of crude lipid extracts into an electrospray ionization source for intrasource separation and identification of numerous lipids, allow for extensive cellular lipid profiles of different tissues being accrued with relative ease. We studied the lipidomic profiles of the breast adipose tissue in lean and DIO normal and tumor bearing mice. Lipidomics analyses were performed using an electrospray triple quadrupole mass spectrometer (TSQ quantum Access Max) and class specific parent-ion or neutral loss scan in positive and negative ion mode with appropriate collision energy. The ratiometic quantification of lipids was done using class specific lipid standards. The phospholipid classes quantified were phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylethanolamine (PE). Our results for the PC class reveal an association between the total carbon chain of the lipids and the lipid concentrations based on four conditions: lean control, obese control, lean tumor bearers, and obese tumor bearers. The highest total carbon chain length is associated with the obese tumor condition. The next highest total carbon chain length is associated with lean tumor condition. This demonstrates that both the presence of the tumor as well as obesity play a role in contributing to a higher number of total carbons in the lipid chains. The other lipid classes analyzed express similar patterns from the data gathered when compared to the PC lipid class. Characterizing a particular lipid signature relevant to breast cancer and obesity may allow its targeting with therapeutic purposes. Citation Format: Osvaldo Perez, Michael Margolis, Ana M. Santander, Mitchell Martinez, Sanjoy Bhattacharya, Marta Torroella-Kouri. Breast cancer and obesity impact the lipid composition of breast adipose tissue: a preliminary study using shotgun lipidomics. [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 3496. doi:10.1158/1538-7445.AM2014-3496</jats:p