Decreased levels of both Stat1 and Stat3 in T lymphocytes from mice bearing mammary tumors.

BACKGROUND: Lymphocytes from tumor-bearing animals have been shown to lack antitumor function. The objective of this study was to investigate the status of the signal transducers, Stat1 and Stat3, in T lymphocytes of animals bearing D1-DMBA-3 mammary tumors and to elucidate if any alterations in these signal transducers can be explained by the presence of tumor-derived factors and correlated with the lack of antitumor function in these cells. MATERIALS AND METHODS: T Lymphocytes from spleens of normal and tumor-bearing mice were purified and assayed for the presence of Stat1 and Stat3 by Western blot analysis. RESULTS: It was found that levels of both Stat1 and Stat3 were reduced in T lymphocytes of tumor-bearers not only in their active, phosphorylated form but in total protein levels. CONCLUSION: These findings indicate that during mammary tumor progression, alteration of various transcription factors may contribute to the down-regulation of immune function.

Antitumor effects of Mucin 1/sec involves the modulation of urokinase-type plasminogen activator and signal transducer and activator of transcription 1 expression in tumor cells.

Expression of the transmembrane isoform of Mucin 1 (MUC1/TM) in an aggressive murine mammary tumor line, DA-3, does not alter tumor development and metastasis, leading to death of the host. However, tumor cells expressing a secreted isoform of MUC1 (MUC1/sec) fail to develop tumors in immunocompetent mice. The rejection of MUC1/sec-expressing tumor cells is immunologically mediated, as, initially, innate cells and, ultimately, T cells are required. After gene array analysis, and confirmation at the protein level, it was discovered that MUC1/sec-expressing tumor cells (DA-3/sec) have a significant reduction in expression of urokinase-type plasminogen activator (uPA) relative to the parental tumor line and tumor cells expressing MUC1/TM. The serine protease uPA has been found to be involved in growth-promoting signaling, angiogenesis, and induction of matrix remodeling leading to metastasis. Although the tumor-promoting Stat3 transcription factor was unaltered in these tumor cells, the tumor-suppressive and IFN-responsive signal transducer and activator of transcription 1 (Stat1) is dramatically up-regulated in DA-3/sec cells. In addition, treatment of various murine and human cell lines with conditioned medium containing MUC1/sec results in up-regulation of Stat1. DA-3/sec tumor cells are also sensitized to the antiproliferative effects of IFN-gamma. Furthermore, transfection of the Stat1 gene into DA-3 tumor cells leads to a down-regulation of uPA and delays tumor progression. Thus, Stat1 up-regulation in DA-3/sec cells seems to play a significant role in the mechanism(s) by which rejection of tumor cells expressing MUC1/sec may be occurring.

The expression of CCL2 by T lymphocytes of mammary tumor bearers: role of tumor-derived factors.

Tumor-associated chemokines, including CC chemokine ligand 2/monocyte chemoattractant protein-1 (CCL2), are thought to play many roles in cancer progression. Here we demonstrate the novel finding that during growth of the D1-7,12-dimethylbenzanthracene-3 mammary tumor in BALB/c mice, there is a dramatic up-regulation of CCL2 in splenic T cells at both the mRNA and protein levels upon stimulation. Of particular relevance is the finding that tumor-infiltrating T cells also produce high levels of CCL2. While a variety of tumor cell lines have been found to produce CCL2, we found no detectable levels of CCL2 protein in supernatants of the cultured mammary tumor cells. Investigation of the mechanisms involved in CCL2 induction showed that treatment of splenic T cells with the tumor-derived factors GM-CSF and phosphatidyl serine (PS) resulted in increased CCL2 production. This increased production may be involved in the downregulation of IFN-gamma by the T cells of tumor-bearing mice previously reported in this model, as treatment of splenic T lymphocytes with CCL2 resulted in a decreased secretion of IFN-gamma by those cells.

A unique mucin immunoenhancing peptide with antitumor properties.

Implantation of DA-3 mammary tumor cells into BALB/c mice results in tumor growth, metastatic lesions, and death. These cells were transfected with genes encoding for either the transmembrane (DA-3/TM) or secreted (DA-3/sec) form of human mucin 1 (MUC1). Although the gene for the secreted form lacks the transmembrane and cytoplasmic domains, the 5' sequences of these mucins are identical; however, the gene for the secreted mucin isoform ends with a sequence encoding for a unique 11 amino acid peptide. The DA-3/TM or DA-3 cells transfected with the neomycin vector only (DA-3/neo) have the same in vivo growth characteristics as the parent cell line. In contrast, DA-3/sec cells fail to grow when implanted in immunocompetent BALB/c animals. DA-3/sec cells implanted in nude mice resulted in tumor development verifying the tumorigenic potential of these cells. Pre-exposure of BALB/c mice to DA-3/sec cells afforded protection against challenge with DA-3/TM or DA-3/neo mammary tumors and the unrelated tumors K7, an osteosarcoma, and RENCA, a renal cell carcinoma. Partial protection against subsequent tumor challenges was also achieved by substituting the 11 amino acid peptide found only in the secreted MUC1 isoform, for the live DA-3/sec cells. Notably, the efficacy of this peptide is not strain restricted because it also retarded the growth of Lewis lung carcinoma cells in C57 BL/6 mice. These findings reveal that a unique peptide present in the secreted MUC1 has immunoenhancing properties and may be a potential agent for use in immunotherapy.

MUC1/sec-expressing tumors are rejected in vivo by a T cell-dependent mechanism and secrete high levels of CCL2.

MUC1/sec is a secreted form of the glycoprotein mucin 1 (MUC1). To characterize the role that MUC1 and MUC1/sec have in tumor progression, these genes were expressed in DA-3 mammary tumor cells. DA-3 cells and DA-3 cells expressing the transmembrane MUC1 gene (DA-3/TM) grow with similar kinetics in BALB/c mice. Surprisingly, DA-3 cells expressing and secreting MUC1/sec (DA-3/sec) fail to form tumors in vivo. The mechanism of rejection was evaluated using mice deficient in constituents of the immune system. All mice lacking IFN-gamma, NK, NKT, or macrophages formed DA-3/sec tumors that regressed shortly after implantation. However, progressively growing DA-3/sec tumors developed in mice devoid of T lymphocytes. The importance of T lymphocytes in the rejection of DA-3/sec tumors was further supported by detection of DA-3-specific CTL in mice challenged with the DA-3/sec tumor. Recruitment of appropriate APC and effector cells is an important first step in the tumor clearance. Indeed, DA-3/sec cells or cell supernatants recruited 3-4 times as many macrophages as DA-3/TM cells in vivo, suggesting that a secreted chemotactic product is produced from DA-3/sec cells. RNA and protein analysis of DA-3/sec cells revealed that several genes are up-regulated by MUC1/sec expression, including MCP-1 (CCL-2). These results suggest DA-3/sec cells are capable of recruiting immune cells, and that rejection of DA-3/sec tumors, although aided by cells of the innate immune response, is ultimately due to T cell-mediated events.

Altered IL-12 Signaling Pathways Contribute to the Deficient IFN-γ Production by T Splenocytes from Tumor-bearing Mice.

IFN-γ is a crucial cytokine produced by T and NK cells. Previous work from our laboratory has reported that in T cells of BALB/c mice bearing the D1-DMBA-3 mammary tumor, IFN-γ production is down-regulated, due to decreased expression of IL-12 by macrophages of tumor bearers. IL-12 is the main inducer of IFN-γ production in T and NK cells. To exert its function, IL-12 interacts with its receptor (IL-12R), activating a JAK/STAT signaling pathway. Our investigation suggests that there is also a deficiency in the response to IL-12 by T cells from tumor hosts. The present work reports the results of RT-PCR experiments in the study of the IL-12R expression on T cells from normal and tumor bearers. Data showed a deficient expression of the IL-12Rß2 chain on T cells from tumor hosts. Gene expression arrays on IL-12-activated T cells from normal and tumor bearers confirmed the RT-PCR results, and also showed decreased expression of IL-18Rα in tumor bearers' T cells. Arrays also showed down-regulated expression of JAK2, STAT 1, 3, 4 and IRF-1. Finally, increased expression of SOCS 1,3,4,5 and 7, as well of Protein inhibitor of activated STATs (Pias) 1 and y was also observed in tumor bearers' T cells.

Up-regulation of matrix metalloproteinase-9 in T lymphocytes of mammary tumor bearers: role of vascular endothelial growth factor.

Matrix metalloproteinase-9 (MMP-9), a matrix-degrading enzyme, is crucial in tumor invasion and metastasis and is implicated in leukocyte extravasation. In this report, we demonstrate that during growth of the D1-7,12-dimethylbenzanthracene-3 mammary tumor in BALB/c mice, there is progressive up-regulation of MMP-9 in splenic T cells at both the transcriptional and translational levels. Our previous work has identified several factors produced by this tumor, including PGE(2), GM-CSF, and phosphatidyl serine; however, none of these agents induces increased production of MMP-9 by normal splenic T cells. Although not produced by the tumor, TNF-alpha and IL-6 are up-regulated in both macrophages and B cells in tumor-bearing mice. Exposure of normal T cells to these two cytokines, however, also fails to up-regulate MMP-9 production. Vascular endothelial growth factor (VEGF) is produced by many tumors, and we determined that the mammary tumor used in our studies expresses high levels of this angiogenic growth factor. Importantly, splenic T cells from tumor bearers constitutively produce increased amounts of VEGF, and treatment of normal T cells with VEGF results in up-regulated MMP-9 production. Of crucial importance is the finding that tumor-infiltrating T cells also produce high levels of VEGF and MMP-9. Our studies indicate that VEGF can act directly on T lymphocytes and that elevated VEGF levels may contribute to the aberrant MMP-9 secretion by mammary tumor bearers' T cells.