Cancer Prevention Research Toxic and Chemopreventive Ligands Preferentially Activate Distinct Aryl Hydrocarbon Receptor Pathways: Implications for Cancer Prevention

The aryl hydrocarbon receptor (AhR) is a ligand-activated regulatory protein that controls estrogen action through two distinct pathways. In one pathway, AhR acts as a transcription factor that induces the expression of the CYP1 family of estrogen-metabolizing genes; in the other pathway, AhR initiates the degradation of the estrogen receptor and suppresses estrogen signaling. The AhR ligand 3,3′-diindolylmethane (DIM) is a beneficial dietary constituent that prevents breast tumors in rodents and is associated with decreased breast cancer risk in humans. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a toxic AhR ligand that is implicated in birth defects, infertility, and cancer. We analyzed MCF-7 cells to gain insight into how two AhR ligands can exert such fundamentally different health effects. We find that DIM and TCDD have differing abilities to activate the distinct AhR-controlled pathways. TCDD strongly induces AhR-dependent CYP1 gene expression, whereas DIM is a relatively weak CYP1 inducer. DIM strongly inhibits estrogen receptor-α expression and estrogen signaling, whereas TCDD has a notably weaker effect on these processes. Small interfering RNA knockdown of AhR confirms that the effects of DIM and TCDD are indeed AhR dependent. Our findings reveal that DIM and TCDD each elicit a unique pattern of change in pathways that control estrogen action; such patterns may determine if an AhR ligand has beneficial or adverse health effects. There is clear evidence that estrogen is associated with breast cancer via two distinct pathways. In one pathway, estrogen metabolites act as carcinogens that damage DNA and initiate breast tumors. In the other pathway, estrogen activates the estrogen receptor (ER), which stimulates the growth of cancerous breast cells (1, 2). The aryl hydrocarbon receptor (AhR), a ligand-activated regulatory protein, controls critical steps in both of these pathways. AhR activates the expression of genes that metabolize estrogen (1, 3) and it initiates the degradation of the ER (4, 5). By controlling these pathways, AhR can influence breast cancer initiation and progression. Epidemiologic studies show that consumption of cruciferous vegetables may provide protection against breast cancer (6). Cruciferous vegetables contain chemopreventive compounds that prevent breast tumors in mice and affect estrogen metabolism and signaling (7–10). Interestingly, these compounds also bind to and activate AhR (11–13). In this study, we compare the AhR-dependent effects of 3,3′-diindolylmethane (DIM), a commercially available nutritional supplement associated with cruciferous vegetables, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a toxic environmental contaminant (14, 15). We find that DIM and TCDD differ in their ability to activate the distinct AhR-controlled pathway, which implies that the biological effects of different AhR ligands may vary considerably. These findings may help to explain how DIM and TCDD can exert such fundamentally different health effects and suggest that AhR may play a role in cancer chemoprotection. Materials and Methods Cells and cell culture MCF-7 cells were obtained from the American Type Culture Collection and cultured as directed. The cells were grown in phenol red–free α-MEM (Invitrogen) supplemented with 5% charcoal/dextran-treated fetal bovine serum (Hyclone) for at least 5 d before harvest. Without this specialized medium, we found that results were inconsistent due to the likely uncontrolled exposure to low levels of endogenous ER-α and AhR ligands. Cells were treated with DIM (Biomol International LP), TCDD (Wellington Laboratories, Inc.), and/or estradiol (E2; Sigma-Aldrich) as indicated. Small interfering RNA treatment A pool of four small interfering RNAs (siRNA) that target AhR or a pool of four nontargeting control siRNAs was purchased from Dharmacon and transfected into cells using DharmaFECT 4 (Dharmacon) as directed by the manufacturer. Cells were harvested and analyzed 72 to 96 h following transfection. Authors' Affiliation: Department of Urology, San Francisco Veterans Affairs Medical Center and the University of California at San Francisco, San Francisco, California Received 7/22/2008; revised 10/9/2008; accepted 11/20/2008; published OnlineFirst 02/17/2009. Grant support: NIH grants RO1CA101844, RO1CA111470, RO1CA108612, RO1AG21418, and T32DK07790 and Veterans Affairs Merit Review. Requests for reprints: Rajvir Dahiya, Department of Urology, San Francisco Veterans Affairs Medical Center and University of California at San Francisco, 4150 Clement Street (112F), San Francisco, CA 94121. Phone: 415-750-6964; Fax: 415-750-6639; E-mail: [email protected]. ©2009 American Association for Cancer Research. doi:10.1158/1940-6207.CAPR-08-0146 OF1 Cancer Prev Res 2009;2(3) March 2009 www.aacrjournals.org Published Online First on February 17, 2009 as 10.1158/1940-6207.CAPR-08-0146 Cancer Research. on June 19, 2017. © 2009 American Association for cancerpreventionresearch.aacrjournals.org Downloaded from Published OnlineFirst February 17, 2009; DOI: 10.1158/1940-6207.CAPR-08-0146