The Cytokine Molecular Mechanisms Section (CMMS) was established in 1986 to develop a research program to elucidate the molecular basis for signal transduction and gene expression regulated by cytokines. At the last site visit we changed fundamental aspects of our research to examine nuclear-receptor (NR) cross-talk with cytokine signal transduction processes. Cytokines have potent effects controlling the proliferation, survival (anti-apoptotic), and drug resistance of a considerably diverse spectrum of tumor cells. Without doubt, the most rigorously studied cytokine which affects these biological processes in a variety of tumor cells, is the pro-inflammatory cytokine IL-6. While there is accumulating basic and clinical research data to suggest that inflammation promotes neoplasia, IL-6 may stand as a centerpiece in this process. Our studies in 2004 are summarized below.1) Molecular cross-talk between nuclear receptors and cytokine signal transduction is involved in the signal pathways of IL-6 in prostate cancer and multiple myeloma. IL-6 was shown to activate the androgen-receptor(AR) by forming a stable STAT3-AR physical complex. This was an important observation demonstrating the non-steroidal activation of AR by an inflammatory cytokine, suggesting inflammation may promote steroid independence of prostate tumors.We have studied the signal and molecular pathways by which IL-6 facilitates the growth and drug resistance in human myeloma cells. We identified the STAT3 pathway as a major pathway involved in the growth of these cells and have blocked this pathway by intervening with two different nuclear receptor mechanisms involving the estrogen receptor and PPAR , each blocking through differing mechanisms. A large number of tumor cells, including prostate, multiple myeloma, renal cell carcinoma, hepatoma, pancreatic carcinoma, etc. secrete and respond to IL-6. The mechanisms are unknown. We have cloned the 2.1. kb human IL-6 promoter, extensively mutated, and studied the regulation of this promoter in autocrine human prostate carcinoma and multiple myeloma cells. We have identified critical transcription factors for each type of cancer cell line that govern the transcriptional regulation and secretion of IL-6. We are currently developing strategies to molecularly block these transcription factors either genetically or pharmacologically to determine the net effects on IL-6 secretion and the biological response of the tumor cell lines in vitro and in vivo.2) Development of new therapeutic strategies for the treatment of breast cancer. Based on experience in studies on the nuclear receptor ER in our laboratory, the focus of our research has been directed toward the development of new intervention strategies for the treatment of breast cancer at the level of ER DNA binding, rather than classical antagonism of estrogen binding. Estrogen receptors (ER) contain zinc fingers motifs required for their activity. We have discovered that certain electrophilic reagents covalently modify ER zinc fingers resulting in the ejection of zinc and selective inactivation of estrogen receptors. These compounds effectively block estrogen-dependent growth of human breast cancer cell lines in vitro and in vivo. Secondly, we have been able to block the transcriptional activity of ER+ MCF-7 and cell growth by utilizing transcription factor decoy (TFD), demonstrating for the first time that TFD can be used against steroid responsive cells. We will continue our studies of novel ER interventions using tamoxifen resistant ER+ cells in vitro and in vivo and the design of more therapeutically efficacious Zn-finger antagonist. 3) IL-6 regulation of epi-genetic gene silencing. We have shown that IL-6 regulates the transcriptional activity of DNA methyltransferase 1 (Dnmt-1). IL-6 activates STAT3, which in turns, induces transcription of the transcription factor Fli-1, which activates the Dnmt-1 promoter. This year we have focused on identifying targeted genes that are silenced via promoter CpG methylation by IL-6 regulation of gene methylation. So far, using human IL-6 responsive myeloma cells, we have determined that IL-6 induces the promoter methylation of two tumor suppressor genes p53 and p16. Four genes known to participate in base excision repair and nucleotide excision repair have been shown to be silenced, leading to defective DNA repair in these cells. Also, MnSOD was shown to be silenced by promoter methylation in human MM cells. We believe that this demonstrates a self destructive untward undesirable or deleterious mechanism of how inflammation may govern tumorigenesis by epigenetic silencing of important repair and tumor suppressor genes. We will further attempt to characterize the mechanisms of genomic silencing by inflammation and, in particular, suppression of DNA repair. Additional emphasis will be aimed at understanding the signal transduction process which activate Dnmt-1 and the mechanisms of how specific genes are selected for genomic silencing.
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