Combination of Inflammatory Si gnaling and an AhR Ligand Leads to Synergistic Induction of IL-6

Research has increasingly shown connections between inflammatory immune responses and carcinogenesis and cancer progression. Evidence points to interleukin 6 (IL-6) differing in its effects by tumor type, in some cancers acting as a positive growth factor, increasing anti-apoptotic signaling, and increasing migration and invasion properties. We are investigating the combinatorial effects of IL-1β and Ah receptor ligands in tumor cell lines and their synergistic induction of IL-6 production. Co-treatment of MCF-7 breast cancer cells and ECC-1 endocervical cancer cells with IL-1β and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 2 hours was shown via quantitative RT-PCR to result in a synergistic induction of IL-6 mRNA transcription and a decrease in CYP1A1 mRNA levels. Quantitative RT-PCR and ELISA assay revealed continuous increases in both mRNA and serum protein levels throughout a 72 hour time course of treatment, with no signs of abating. Additional AhR ligands, including benzo[a]pyrene, similarly show synergistic activity in conjunction with IL-1β, with regard to IL-6 induction. Although the promoters for IL-6 and IL-8 have marked similarities in containing NFκB, C/EBPβ and c-Jun regulatory binding sites, IL-8 transcription does not follow the pattern of synergistic increase seen with IL-6. Therefore, the combination of pro-inflammatory IL-1β and AhR ligands shows a context-specific ability to up-regulate cellular IL-6 output for a sustained period of time. This event could lead to instigation of both autocrine and paracrine loop signaling within the tumor microenvironment. These results would suggest a mechanism that could in part explain the tumor promoting properties of certain AhR ligands (e.g benzo[a]pyrene). Introduction The AHR has often been studied through its role in mediating the toxicity of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD), commonly referred to as dioxin. This persistent environmental contaminant bioaccumulates in the food chain and is essentially not metabolized by mammals. TCDD has been shown to be a carcinogen by way of AhR binding, which leads to heterodimerization with ARNT and transcriptional activation of drug metabolizing enzymes, such as CYP1A1 (1). TCDD has also been shown to be a potent tumor promoter in various models, though the precise mechanism(s) of AhRmediated tumor promotion are largely unknown. The AhR is known to functionally interact with numerous transcription factor pathways, and various reports have demonstrated “cross-talk” between the immunologically relevant estrogen receptor (ER) or NF-κB transcription factors, and the AhR (2). Whether the Ah receptor can modulate expression of key NF-κB target genes such as IL-6 is poorly understood. IL-6 is a pleiotropic cytokine “classically” involved in the acute phase response and lymphocyte differentiation and proliferation following inflammatory stimuli, as well as being found in various types of non-immune tissues. Additionally, IL-6 expression in tumor cells is known to elicit both proand anti-tumorigenic properties. Studies have shown both proand anti-proliferative effects of IL-6 on breast cancer cells lines, with the stage of carcinoma and estrogen receptor status playing a role. Supernatants taken from multidrug resistant breast carcinoma cell lines show markedly higher IL-6 levels than those of chemo-sensitive parental cells (3). Numerous clinical studies have shown that cervical cancer leads to elevated IL-6 levels and elevated IL-6 gene expression has been correlated with the presence of invasive cervical carcinomas (4). However, IL-6 expression in breast cancer has been implicated to correlate both positively and negatively with overall prognosis (3). Low IL-6 levels have been observed in certain established breast cancer cell lines, such as MCF-7 cells (5). The estrogen receptor has also been shown to repress IL-6 transcription, thus lending credence to notions of receptor cross-talk and a rationale for the higher IL-6 expression in invasive estrogen receptor negative breast cancer cell lines. For the first time, we show that an AhR ligand can synergize with cytokines to enhance Il-6 production in some tumor cell lines Conclusions • Co-treatment of breast cancer and endocervical cancer cell lines with TCDD and IL-1β results in synergistic increases of IL-6 transcription and lowered Cyp1A1 transcription • The synergistic increase in IL-6 transcription is comparable to an increase in protein secretion, continuing through 72 h • Conditioned media from activated monocytes is sufficient to result in synergistic IL-6 induction with TCDD, providing evidence of the possibility of this effect occurring within a tumor microenvironment • IL-8, while regulated similarly to IL-6, is not transcriptionally affected by the addition of an AhR ligand References 1. Schwarz M, Appel KE. Carcinogenic risks of dioxin: mechanistic considerations. Regul Toxicol Pharmacol. 2005 Oct;43(1):19-34. 2. Tian Y, Rabson AB, Gallo MA. Ah receptor and NF-kappaB interactions: mechanisms and physiological implications. Chem Biol Interact 2002;141(12):97-115. 3. Knupfer H, Preiss R. Significance of interleukin-6 (IL-6) in breast cancer. Breast Cancer Res Treat. 2007 Apr;102(2):129-135. 4. Takano H, et al. Interleukin-6 (IL-6) production in carcinoma of the cervix. Arch Gynecol Obstet. 1996;258(1):25-33. 5. Sasser AK, et al. Interleukin-6 is a potent growth factor for ER-alpha-positive human breast cancer. Faseb J 2007;21(13):3763-70. Implication Scheme Potential Outcomes Control IL-1β TCDD TCDD + IL-1β 0 3 6 9 12 15 IL -6 m R N A L ev el s Control IL-1β TCDD TCDD + IL-1β 0 10 20 30 40 C yp 1a 1 m R N A L ev el s Fig 1. Co-treatment with TCDD and IL-1β results in synergistic IL-6 transcription, while IL-1β decreases Cyp1A1 transcription in MCF-7 cancer cells. A & B, MCF-7 cells were plated in 6-well dishes, serum starved for 24 h, and treated for 2 h with DMSO vehicle, 10 ng/ml IL-1β, 1 nM TCDD, or TCDD and IL-1β. IL-6 and Cyp1a1 mRNA levels were determined by quantitative real-time PCR. Control IL-1β TCDD TCDD + IL-1β 0.0 0.5 1.0 1.5 2.0 2.5 IL -6 m R N A L ev el s Control IL-1β TCDD TCDD + IL-1β 0 2 4 6 C yp 1a 1 m R N A L ev el s Fig 2. Cyp1a1 and IL-6 regulation by combinatorial TCDD and IL-1β treatment is also evident in the ECC-1 endocervical cancer cell line. A & B, ECC-1 cells were plated in 6-well dishes, serum starved for 24 h, and treated for 2 h with DMSO vehicle, 10 ng/ml IL-1β, 1 nM TCDD, or TCDD and IL-1β. IL-6 and Cyp1a1 mRNA levels were determined by quantitative real-time PCR. 24 48 72 24 48 72 24 48 72 24 48 72 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 * Control IL-1β TCDD TCDD+ IL-1β Hours Post-treatment IL -6 p ro te in le ve ls