Christoph Vogel
email: cfvogel@ucdavis.edu

Gwendal Lazennec

Sarah Y Kado

Carla Dahlem

Yi He

Alejandro Castaneda

Yasuhiro Ishihara

Christian Vogeley

Andrea Rossi

Thomas Haarmann-Stemmann

Christoph Fa Vogel

Thomas Haarmann- Stemmann

Juliann Jugan

Hidetoshi Mori

Alexander D Borowsky

La Merrill

Colleen Sweeney

Targeting the aryl hydrocarbon receptor signaling pathway in breast cancer development

Keywords: AhR, AhRR, carcinogenicity, breast cancer, C/EBPβ, cyclooxygenase 2, inflammation

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Introduction

Environmental exposure to toxicants including dioxins and many other dioxin-like compounds (DLC) which bind to the aryl hydrocarbon receptor (AhR) and activate the AhR signaling pathway, is associated with the development of malignancies including breast cancer [START_REF] Narasimhan | Towards Resolving the Pro-and Anti-Tumor Effects of the Aryl Hydrocarbon Receptor[END_REF][START_REF] Kolluri | Role of the aryl hydrocarbon receptor in carcinogenesis and potential as an anti-cancer drug target[END_REF][START_REF] Murray | Aryl hydrocarbon receptor ligands in cancer: friend and foe[END_REF][START_REF] Warner | Dioxin exposure and cancer risk in the Seveso Women's Health Study[END_REF]. Indeed, due in part to the extensive mechanistic information indicating that the DLCs act through a mechanism involving the AhR the International Agency for Research on Carcinogens named DLCs as "carcinogenic to humans" (Group 1) [START_REF] Steenland | Dioxin revisited: developments since the 1997 IARC classification of dioxin as a human carcinogen[END_REF]. The activated AhR pathway results in changes of the expression profile of cytokines and immune modulatory enzymes which may contribute to the carcinogenic effects of AhR-activating toxicants [START_REF] Murray | Aryl hydrocarbon receptor ligands in cancer: friend and foe[END_REF]. The repressor protein of the AhR (AhRR) has been found to suppress the canonical AhR signaling pathway as well as the activation of inflammatory cytokines [START_REF] Hahn | Regulation of constitutive and inducible AHR signaling: complex interactions involving the AHR repressor[END_REF][START_REF] Vogel | Transgenic Overexpression of Aryl Hydrocarbon Receptor Repressor (AhRR) and AhR-Mediated Induction of CYP1A1, Cytokines, and Acute Toxicity[END_REF]. Moreover, reports have shown that the AhRR suppresses growth of tumor cells including breast cancer cells in vitro [START_REF] Zudaire | The aryl hydrocarbon receptor repressor is a putative tumor suppressor gene in multiple human cancers[END_REF]. Support for the premise comes from a recent report, showing that breast cancer patients who had low AhRR expression also had shorter metastasis-free survival and identified AhRR as an independent prognostic factor [START_REF] Vacher | High AHR expression in breast tumors correlates with expression of genes from several signaling pathways namely inflammation and endogenous tryptophan metabolism[END_REF]. Literature also indicates that the AhR regulates normal development of the mammary gland [START_REF] Lew | Activation of the Aryl Hydrocarbon Receptor During Pregnancy in the Mouse Alters Mammary Development Through Direct Effects on Stromal and Epithelial Tissues[END_REF][START_REF] Vorderstrasse | A novel effect of dioxin: exposure during pregnancy severely impairs mammary gland differentiation[END_REF][START_REF] Belton | Metabolomics Reveals Aryl Hydrocarbon Receptor Activation Induces Liver and Mammary Gland Metabolic Dysfunction in Lactating Mice[END_REF] revealing this tissue as a sensitive target of environmental pollutants containing AhR activating chemicals.

The finding of overexpressed AhR in mammary cancer in rats [START_REF] Trombino | Expression of the aryl hydrocarbon receptor/transcription factor (AhR) and AhR-regulated CYP1 gene transcripts in a rat model of mammary tumorigenesis[END_REF] raised the question of whether AhR is involved in breast cancer progression. Indeed, several in vitro studies demonstrated the contribution of AhR to carcinogenic progression [START_REF] Wang | The diverse and important contribution of AHR to cancer and cancer immunity[END_REF]. For instance, Brooks and Eltom [START_REF] Brooks | Malignant transformation of mammary epithelial cells by ectopic overexpression of the aryl hydrocarbon receptor[END_REF] showed that overexpression of AhR in human mammary epithelial cells led to cellular transformation and epithelial to mesenchymal transition (EMT). Work from our group revealed that chronic exposure of human breast MCF10AT1 and MCF-7 cells to estradiol (E2) resulted in AhR overexpression and downregulation of estrogen receptor alpha (ERα) and progesterone receptor [START_REF] Zou | Long-term exposure to beta-hexachlorocyclohexane (beta-HCH) promotes transformation and invasiveness of MCF-7 human breast cancer cells. Long-term exposure to beta-hexachlorocyclohexane (beta-HCH) promotes transformation and invasiveness of MCF-7 human breast cancer cells[END_REF][START_REF] Wong | Characterization of MCF mammary epithelial cells overexpressing the Aryl hydrocarbon receptor (AhR)[END_REF]. Both cell lines exhibited increased proliferation, matrigel invasion, and apoptosis resistance compared to control cells. More recently, we and other groups found that the AhR is frequently overexpressed in human breast cancer, particularly ER-negative breast cancer [START_REF] Vacher | High AHR expression in breast tumors correlates with expression of genes from several signaling pathways namely inflammation and endogenous tryptophan metabolism[END_REF][START_REF] Amato | A TDO2-AhR signaling axis facilitates anoikis resistance and metastasis in triple-negative breast cancer[END_REF][START_REF] Vogel | Interaction of aryl hydrocarbon receptor and NF-κB subunit RelB in breast cancer is associated with interleukin-8 overexpression[END_REF]. AhR overexpression in this setting is closely associated with elevated expression of the NF-кB subunit RelB and the inflammatory markers IL-8 (CXCL1 in mouse) and COX-2 [START_REF] Vogel | Interaction of aryl hydrocarbon receptor and NF-κB subunit RelB in breast cancer is associated with interleukin-8 overexpression[END_REF]. Interestingly, COX-2 and chemokines such as CXCL1, CXCL5, and the chemokine receptor CXCR2 have been identified as critical genes that mediate breast cancer metastasis to lung, lymph nodes, and bone [START_REF] Minn | Genes that mediate breast cancer metastasis to lung[END_REF][START_REF] Romero-Moreno | The CXCL5/CXCR2 axis is sufficient to promote breast cancer colonization during bone metastasis[END_REF][START_REF] Boissière-Michot | Prognostic Value of CXCR2 in Breast Cancer[END_REF]. A recent genome wide analysis of AhR and AhRR binding found a significant overlap in sequences binding both proteins, suggesting that AhRR most likely functions as a tumor suppressor by opposing AhR-driven gene expression [START_REF] Yang | Genome-wide mapping and analysis of aryl hydrocarbon receptor (AHR)-and aryl hydrocarbon receptor repressor (AHRR)-binding sites in human breast cancer cells[END_REF].

Despite dysregulation of the AhR/AhRR axis in breast cancer, relatively little is known about the function of AhRR in vivo [START_REF] Hahn | Regulation of constitutive and inducible AHR signaling: complex interactions involving the AHR repressor[END_REF][START_REF] Vogel | The aryl hydrocarbon receptor repressor -More than a simple feedback inhibitor of AhR signaling: Clues for its role in inflammation and cancer[END_REF]. In the current study we have examined several AhR-driven outcomes, to determine whether AhRR functionally opposes AhR and is able to suppress the development of mammary tumors. Using our previously characterized AhRR transgenic mouse, we demonstrate that AhRR overexpression restricts the growth of both E0771 mammary tumor cells and mammary tumors in the Polyoma Middle T antigen (PyMT) model of mammary tumorigenesis. Furthermore, the tumor suppressive function of AhRR was confirmed in mouse PyMT-derived mammary tumor cells and human breast cancer cell lines indicating that AhRR inhibits cell proliferation and AhR-mediated apoptotic resistance.

Materials and Methods

Reagents

Dimethyl sulfoxide (DMSO) was purchased from Sigma. [γ- 32 P]ATP (6000 Ci/mmol) was provided by ICN Biochemicals, Inc. (Costa Mesa, CA, USA). 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (>99% purity) was originally obtained from Dow Chemical Co. (Midland, MI, USA). Other molecular biological reagents were purchased from Cayman Chemicals (Ann Arbor, MI, USA) and Applied Biosystems (Foster City, CA, USA).

Cell Culture and Transfection Experiments

Mammary epithelial cells (UCD-PYMT) were isolated from the mammary tumor of a 26-week-old B6.FVBTg(MMTV PyVT)634Mul/LellJ (PyMT) hemizygous mouse (Jackson Laboratory, Bar Harbor, ME) [START_REF] Borowsky | Syngeneic mouse mammary carcinoma cell lines: two closely related cell lines with divergent metastatic behavior[END_REF][START_REF] Davie | Effects of FVB/NJ and C57Bl/6J strain backgrounds on mammary tumor phenotype in inducible nitric oxide synthase deficient mice[END_REF] as described in Pénzváltó et al [START_REF] Pénzváltó | A Syngeneic ErbB2 Mammary Cancer Model for Preclinical Immunotherapy Trials[END_REF]. Briefly, the mammary tumor was washed twice in PBS (Invitrogen, Carlsbad, CA) before it was mechanically dissociated and minced in a solution of serumfree DMEM:F12 (Invitrogen) with HEPES (Invitrogen), supplemented with 0.5 mg/ml Penicillin/Streptomycin (Invitrogen), 2% bovine serum albumin fraction V (Invitrogen), 5 μg/ml insulin (Sigma Aldrich, Saint Louis, MO), 10 ng/ml cholera toxin (Sigma Aldrich), and 3 mg/ml collagenase (Worthington Biochemical Corp., Lakewood, NJ). The tissue was then digested with gentile agitation overnight at room temperature before differential centrifugation at 80x g for 1.5 minutes. The remaining cell pellet of UCD-PYMT cells was washed in DMEM:F12 (Invitrogen) and centrifuged at 80x g for 4 minutes before being cultured in Advanced DMEM/F12 culture medium (Gibco) supplemented with 5% FBS, 1% ITS Premix (Corning, Concord, NC), 0.5 mg/ml Penicillin/Streptomycin (Invitrogen), and 1% GlutaMax (Gibco) as described [START_REF] Borowsky | Syngeneic mouse mammary carcinoma cell lines: two closely related cell lines with divergent metastatic behavior[END_REF][START_REF] Davie | Effects of FVB/NJ and C57Bl/6J strain backgrounds on mammary tumor phenotype in inducible nitric oxide synthase deficient mice[END_REF]. UCD-PYMT epithelial origin was further confirmed by 100% E-cadherin positive staining of cells (DAPI positive) at 24h and after 5 days in culture (Supplementary Figure 1). UCD-PYMT were transiently transfected with a cDNA mouse AhRR expression plasmid or an A-C/EBP vector that produce dominant-negative proteins that specifically inhibit the DNA binding of the C/EBP members kindly provided by Charles Vinson (NCI, Bethesda, MD, USA). Transient transfection was performed using jetPEI (PolyTransfection; Qbiogene, Irvine, CA, USA), according to the manufacturer's instructions. The transfection was allowed to proceed for 16 h, and cells were treated with 1 nM TCDD or 0.1% DMSO (control) for 24 h before induction of apoptosis or treatment with TCDD for RNA expression analysis. For DRE luciferase reporter assay UCD-PYMT cells were transiently transfected with a DRE reporter plasmid. After 16 h cells were treated with 1 nM TCDD or 0.1% DMSO (control) for 4 h. Cells were lysed and luciferase activity was measured with the Luciferase Reporter Assay System (Promega Corp., Madison, WI) using a luminometer (Berthold Lumat LB9501/16; Pittsburg, PA). Relative light units were normalized to protein concentration using Bradford dye assay (Bio-Rad Laboratories, Inc., Hercules, CA).

MDA-MB 231 and MCF-7 cells were cultured in DMEM plus 10 % FCS. Cells were seeded in 12well plates at 2x^10 5 cells/well. After 24 h cells were transfected with a rat AhRR expression plasmid, which was generously provided by Yoshio Inouye. Control cells were transfected with the empty vector.

Apoptosis was induced with Etoposide (50 µM) and Doxorubicin (10 µM) (Cayman Chemical, Ann Arbor, MI) and control cells received 0.1 % DMSO vehicle.

Generation of CRISPR/Cas9 AhR mutants of MDA-MB 231 and MCF-7 cells

A gRNA targeting AhR exon 2 (5'-AAGTCGGTCTCTATGCCGCTTGG-3') was designed using the CRISPR design tool CHOPCHOP (http://chopchop.cbu.uib.no/) and cloned into a modified version of the PX458 plasmid available on Addgene (48138). The resulting bicistronic vector encoded the gRNA and the Cas9 nuclease. gRNA activity and efficiency were assessed using High Resolution Melt Analysis (HRMA) (28) using the following primers: fw 5'-GCCAATCCCAGCTGAAGG-3' rv 5'-TAGCCAAACGGTCCAACTCT-3' and a MyGo PRO real time PCR (IT-IS Life Science Ltd). MDA-MB 231 and MCF-7 cells were transfected with nuclease plasmids in antibiotic-free medium in a 12-well plate using FuGENE HD (Roche) according to the manufacturer's protocol. After 48 hours cells were sorted (FACS) and plated as single cells in a 96-well plate and duplicated after a week. Clones were lysed in Proteinase-K and genotyped using high-resolution melt analysis and SANGER sequencing.

AhR knockout in MCF-7 and MDA-MB 231 cells was confirmed in Western blot analysis (Fig. 6E andF).

Mice and Treatment

The [START_REF] Sugiura | Studies in a tumor spectrum[END_REF]. The E0771 cell line is a spontaneously developing medullary breast adenocarcinoma from C57BL/6 mice [START_REF] Johnston | Functional and molecular characterization of E0771.LMB tumors, a new C57BL/6-mouse-derived model of spontaneously metastatic mammary cancer[END_REF]. The cultured E0771 tumor cell suspension was resuspended in PBS to obtain the desired concentration of 5.0 x 10 6 cells/mL. A 1 mL syringe affixed with a 23-G needle was loaded with 0.1 mL of the E0771 tumor cell suspension (500,000 cells). For control, 0.1 mL PBS alone was injected. E0771 cells were injected subcutaneously into the fourth inguinal mammary gland of wt and AhRR Tg mice (10 weeks old, 10 female mice in each group). 24 h after injection of E0771 cells, mice were treated with vehicle (corn oil or PBS) or TCDD (10 μg/kg bw) in order to test possible enhancing effects of TCDD on tumor growth of E0771 breast cancer cells in wt and AhRR Tg mice. TCDD was administered via intraperitoneal (i.p.) injection. Each mouse was palpated three times a week at the injection site and the tumor size was measured using a slide microcaliper for 18 days post-injection.

These data were used to determine the tumor volume by employing the following formula V = (L*W*H)/2.

Virgin mammary glands and lungs from 5-month-old PyMT/wt and PyMT/AhRR + transgenic female mice were prepared at necropsy for histology. Whole mounts were spread on slides, fixed and stained with hematoxylin in order to elucidate ductal structure as described (32).

Electrophoretic Mobility Shift Assay (EMSA)

Nuclear extracts were isolated from UCD-PYMT as described previously [START_REF] Vogel | Cross-talk between aryl hydrocarbon receptor and the inflammatory response: a role for nuclear factor-κB[END_REF]. UCD-PYMT were treated with TCDD for 90 min and harvested in ice cold Dulbecco's PBS. The DNA/protein binding reactions were carried out in a total volume of 15 μL containing 10 μg of nuclear protein, 60,000 cpm of double-stranded C/EBP consensus oligonucleotide (5′-TGCAGATTGCGCAATCTGCA-3′) plus 1 μg of poly(dI•dC). The samples were incubated at room temperature for 20 min. Competition experiments were performed in the presence of a 100-fold molar excess of unlabeled oligo. Protein-DNA complexes were resolved on a nondenaturating polyacrylamide gel and visualized by exposure of the dried gels to x-ray films. Protein-DNA complexes were quantified using ChemImager TM 4400 (Alpha Innotech Corp.).

RNA Isolation and Real-Time PCR

Total RNA was isolated from cells using a Quick-RNA Mini prep isolation kit (Zymo Research), and cDNA synthesis was performed as described (33) using a cDNA synthesis kit Applied Biosystems (Foster City, CA, USA). Detection of β-actin and differentially expressed target genes was performed with a LightCycler LC480 Instrument (Roche Diagnostics, Indianapolis, IN, USA) using the Fast SYBR Green Master Mix (Applied Biosystems) according to the manufacturer's instructions. The primers for each gene were designed on the basis of the respective cDNA or mRNA sequences using OLIGO primer analysis software provided by Steve Rozen and the Whitehead Institute/Massachusetts Institute of Technology Center for Genome Research so that the targets were 100-200 bp in length. PCR amplification was carried as described [START_REF] Vogel | Cross-talk between aryl hydrocarbon receptor and the inflammatory response: a role for nuclear factor-κB[END_REF]. To confirm the amplification specificity, the PCR products were subjected to melting curve analysis.

Western blotting

Proteins from mouse tissue samples were isolated and prepared for Western blot as described [START_REF] Vogel | Transgenic Overexpression of Aryl Hydrocarbon Receptor Repressor (AhRR) and AhR-Mediated Induction of CYP1A1, Cytokines, and Acute Toxicity[END_REF].

Cells were collected and lysed with radioimmunoprecipitation assay buffer and equal amounts of protein were loaded, separated via SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The blocked membranes were incubated with the specific antibodies. The antibodies against actin and human AhR were purchased from Cell Signaling Technologies (Danvers, MA, USA), while the purified rabbit anti-AhRR antibody was purchased from Novoprotein (Fremont CA, USA) and mouse AhR purchased from Enzo (Farmingdale, NY, USA). Bands were visualized using peroxide substrates (SuperSignal West Pico, ThermoFisher Scientific, USA) after incubation with a peroxideconjugated antibody. The band intensity was quantified using ChemImager TM 4400 (Alpha Innotech Corp.). reagent was added to the designated wells. After a 4 h incubation, the MTT formazan precipitate was dissolved in DMSO and the absorbance was determined at 490 nm using a plate reader (Berthold, USA).

Cell growth

UCD-PYMT cells and

Apoptosis Assay

UCD-PYMT cells (5 × 10 5 cells) were seeded in a 6 cm dish and exposed to TCDD for 24 h prior to apoptosis induced by Etoposide and Doxorubicin and detected by Annexin V staining as described previously [START_REF] Bekki | The aryl hydrocarbon receptor (AhR) mediates resistance to apoptosis induced in breast cancer cells[END_REF]. The detection of the phosphorylated form of variant histone H2AX (γ-H2AX), which occurs specifically at sites of DNA double-strand breaks was used to determine apoptotic cells via flow cytometry in MDA-MB 231 and MCF-7 cells. Cells were seeded in 12-well plates at 2x10 5 cells/well.

For FACS analyses the supernatant was collected and the cell layer was washed with PBS and trypsinized. Trypsinized cells were collected and transferred to the respective supernatant. Cells were centrifuged at 300 g for 5 min at room temperature, followed by washing with PBS. Pellets were resuspended in 300 µl fluorochrome solution containing 0.1 % sodium citrate, 0.1 % Triton X-100, 50 µg/ml propidium iodide and 25 ng APC anti-γH2AX (Ser139) antibody (BioLegend, San Diego, CA, USA). After incubation in the dark for 20 min, cells were analyzed by flow cytometry. The acquired data were analyzed using the FlowJo software package (Tree Star Inc., Ashland, OR, USA).

Statistical Analysis

All experiments were repeated a minimum of three times, and data were expressed as mean ± S.D. Differences were considered significant at p < 0.05. A comparison of two groups was made with an unpaired, two-tailed Student's t test. A comparison of multiple groups was made with analysis of variance followed by a Dunnett's or Tukey's test.

Results

Suppression of Tumor Growth in AhRR Tg Mice. To examine the tumor-suppressive action of

AhRR in vivo, we compared growth of syngeneic E0771 mammary tumor cells in the mammary fat pad of wildtype (wt) B6 and AhRR Tg mice. The E0771 cell line is a spontaneously developing medullary breast adenocarcinoma derived from C57BL/6 mice and a model of triple negative breast cancer (TNBC). AhRR Tg mice were created and previously characterized by our group and exhibit overexpression of AhRR in all tissues examined [START_REF] Vogel | Transgenic Overexpression of Aryl Hydrocarbon Receptor Repressor (AhRR) and AhR-Mediated Induction of CYP1A1, Cytokines, and Acute Toxicity[END_REF]. Results indicate a significantly suppressed growth of E0771 mammary tumor cells in AhRR Tg mice compared to wt mice (Fig. 1A). Tumor growth was significantly enhanced after TCDD treatment only in wt mice while still suppressed in AhRR Tg mice (Fig. 1B). TCDD is a prototypical ligand of AhR and the most toxic congener of dioxins. These data indicate that the overexpression of AhRR in the host environment is sufficient to suppress AhR-driven mammary tumor growth. We next generated PyMT/AhRR + mice and followed tumor growth over time. As shown in figure 3, AhRR overexpression (PyMT/AhRR + ) has a significant impact on tumor kinetics, increasing time to palpable tumor onset and decreasing incidence by the study censor date (Fig. 3A). AhRR overexpression also decreased the number of palpable tumors at necropsy and reduced tumor multiplicity (Fig. 3B andC). Expression analysis confirmed that AhR as well as COX-2 and C/EBPβ were suppressed in mammary tumors of PyMT/AhRR + mice compared to PyMT/wt mice (Fig. 3 D to H). Representative whole mounts of mammary glands from 5 months old PyMT/wt and tumor free PyMT/AhRR + mice are shown in figures 4 A and B. As expected, multi-focal mammary tumors are evident in PyMT/wt mice 5 months after birth. Furthermore, while metastatic colonies were evident in whole mounts of lungs from PyMT/wt mice (Fig. 4C), PyMT/AhRR + mice showed no evidence of lung metastasis 5 months after birth. (Fig. 4D). At necropsy 60% of PyMT/AhRR + mice were devoid of lung metastasis, whereas all PyMT/wt mice developed metastatic foci in lung at necropsy (Fig. 4E).

AhRR Suppresses AhR-induced expression of inflammatory markers

To further examine the effect of AhRR on the expression of COX-2 and C/EBPβ and to test mammary tumor cell-intrinsic effects of AhRR overexpression, we utilized UCD-PYMT cells, a mammary tumor cell line previously established from PyMT mice. UCD-PYMT cells were transfected with control plasmid or plasmid expressing AhRR and treated with 1 nM TCDD to engage AhR signaling. After 24 hours, the expression of both C/EBPβ and COX-2 was induced by TCDD (Figure 5A andB). Notably, this induction was significantly restricted, for both C/EBPβ and COX-2 in UCD-PYMT overexpressing AhRR (Figure 5C). As reported earlier, the TCDD-mediated induction of COX-2 may involve the activation of PKA and DNA binding of C/EBPβ [START_REF] Vogel | Regulation of prostaglandin endoperoxide H synthase-2 induction by dioxin in rat hepatocytes: possible c-Src-mediated pathway[END_REF][START_REF] Vogel | Dioxin increases C/EBPbeta transcription by activating cAMP/protein kinase A[END_REF]. Therefore, DNA binding activity of C/EBPβ was determined utilizing EMSA with nuclear proteins prepared from control and AhRRtransfected UCD-PYMT cells. TCDD stimulated DNA binding to a C/EBP consensus element in both cases but this binding was significantly decreased in control and TCDD-treated AhRR overexpressing cells (Fig. 5D andE). Furthermore, we found that AhRR reduced the basal as well as TCDD-induced activity of AhR in UCD-PYMT cells (Fig. 5F) indicating the presence of endogenous ligands causing an increased constitutive level of AhR activity in these cells.

AhRR overexpression inhibits cell growth and sensitizes UCD-PYMT and MDA-MB 231 and MCF-7 cells to apoptosis induced by anti-cancer drugs.

A hallmark of neoplastic development is deregulated cell proliferation and resistance to apoptosis.

Previous reports have shown that AhRR inhibits cell growth and resistance to apoptotic signals in human breast epithelial and cancer cells [START_REF] Zudaire | The aryl hydrocarbon receptor repressor is a putative tumor suppressor gene in multiple human cancers[END_REF][START_REF] Wong | Characterization of MCF mammary epithelial cells overexpressing the Aryl hydrocarbon receptor (AhR)[END_REF][START_REF] Kanno | The inhibitory effect of aryl hydrocarbon receptor repressor (AhRR) on the growth of human breast cancer MCF-7 cells[END_REF]. Here we tested the effect of AhRR on cell growth in UCD-PYMT cells after transfection with a mouse AhRR expression plasmid (Fig. 6A). Cell proliferation rate was monitored from day 1 through day 4. The results show that AhRR significantly reduced the growth of UCD-PYMT cells compared to control cells (Fig. 6A). In order to test if C/EBP binding plays a role in the inhibitory effect on cell growth we transfected cells with a vector expressing dominant negative proteins to block DNA binding of C/EBP proteins. The results show that A-C/EBP inhibits the growth of UCD-PYMT cells similar to overxpression of AhRR.

Given our prior findings that AhR signaling mediates breast cancer cell resistance to apoptosis induced by UV radiation or anti-cancer drugs (34), we next examined the impact of AhRR expression on response of UCD-PYMT cells to doxorubicin (Dox), a DNA intercalating agent and etoposide (EtOP), an inhibitor of Topoisomerase-II. Treatment of UCD-PYMT cells with either Dox or EtOP led to a significant increase in apoptosis which was rescued in both cases by TCDD/AhR signaling (Fig. 6B). for DSB [START_REF] Rogakou | DNA doublestranded breaks induce histone H2AX phosphorylation on serine 139[END_REF]. In fact, AhR deficiency as well as AhRR overexpression resulted in an accumulation of these DNA lesions in both breast cancer cell-lines (Fig. 7B andD), suggesting a regulatory function of AhR in DSB repair. Western Blot data and mRNA expression analysis confirm the successful knockout of AhR in MCF-7 and MDA-MB 231 breast cancer cell lines (Fig. 7E andF).

More apoptosis was observed in

Discussion

In prior work, we demonstrated that overexpression of AhRR in vitro, in human breast cancer cells, inhibits cell survival mediated by AhR [START_REF] Wong | Characterization of MCF mammary epithelial cells overexpressing the Aryl hydrocarbon receptor (AhR)[END_REF]. This study is the first to demonstrate that AhRR overexpression restricts mammary tumor cell growth and tumorigenesis in vivo. In the syngeneic E0771 model, we demonstrate that AhRR overexpression inhibits basal and AhR-driven (TCDDstimulated) mammary tumor cell growth. The results suggest that AhRR overexpression in the host environment is sufficient to inhibit orthotopic growth of mammary tumor cells. This builds on our prior study in which we demonstrated that growth of lymphoma cells was suppressed in AhRR Tg mice [START_REF] Vogel | A Protective Role of Aryl Hydrocarbon Receptor Repressor in Inflammation and Tumor Growth[END_REF]. We note that the growth of E0771 cells was suppressed in untreated AhRR transgenic mice suggesting that AhRR overexpression in the host may suppress tumor growth independent of exogenous and toxic AhR ligands. Interestingly, a recent report using immortalized mouse mammary gland fibroblasts showed that knockout of AhR also reduced the potential to induce tumors in a mouse xenograft model [START_REF] Mulero-Navarro | Immortalized mouse mammary fibroblasts lacking dioxin receptor have impaired tumorigenicity in a subcutaneous mouse xenograft model[END_REF] indicating that suppression of AhR by AhRR as well as deficiency of AhR impairs tumorigenicity. One possibility is that the increased expression and activity of AhR found in breast tumor cells causes altered levels of tryptophan metabolizing enzymes as shown for indoleamine 2,3-dioxygenase (IDO) and tryptophan-2,3-dioxygenase (TDO) [START_REF] Bekki | The aryl hydrocarbon receptor (AhR) mediates resistance to apoptosis induced in breast cancer cells[END_REF][START_REF] Vogel | Aryl hydrocarbon receptor signaling mediates expression of indoleamine 2,3-dioxygenase[END_REF][START_REF] Novikov | An Aryl Hydrocarbon Receptor-Mediated Amplification Loop That Enforces Cell Migration in ER-/PR-/Her2-Human Breast Cancer Cells[END_REF] and generate high levels of the endogenous AhR ligand kynurenine (Kyn). C/EBPβ as well as COX-2 have recently been found to maintain the constitutive expression of Kyn-producing TDO in human glioblastoma [START_REF] Kudo | Constitutive Expression of the Immunosuppressive Tryptophan Dioxygenase TDO2 in Glioblastoma Is Driven by the Transcription Factor C/EBPbeta[END_REF][START_REF] Ochs | Tryptophan-2,3-dioxygenase is regulated by prostaglandin E2 in malignant glioma via a positive signaling loop involving prostaglandin E receptor-4[END_REF], suggesting that this transcription factor and COX-2 critically shape the protumorigenic properties of AhR. The IDO-Kyn-AhR signaling pathway has been shown to mediate immunosuppression involving Tregs and tumor-associated macrophages, which can be reversed by AhR inhibition [START_REF] Campesato | Blockade of the AHR restricts a Treg-macrophage suppressive axis induced by L-Kynurenine[END_REF]. Interestingly, a recent meta-analysis across military and civilian cohorts indicates that lower AhRR methylation correlates with lower levels of Kyn [START_REF] Smith | Epigenome-wide meta-analysis of PTSD across 10 military and civilian cohorts identifies methylation changes in AHRR[END_REF] suggesting that higher AhRR activity may regulate the level of Kyn. Consequently, the IDO-Kyn-AhR signaling pathway provides a new target in cancer immunotherapy as discussed recently [START_REF] Opitz | An endogenous tumour-promoting ligand of the human aryl hydrocarbon receptor[END_REF] and AhRR may provide an important tool to inhibit this pathway (Fig. 8).

The PyMT mammary tumorigenesis model is a well-characterized and widely used model of ERnegative, metastatic breast cancer. We find that expression changes in the AhR/AhRR axis observed in human breast cancer [START_REF] Vacher | High AHR expression in breast tumors correlates with expression of genes from several signaling pathways namely inflammation and endogenous tryptophan metabolism[END_REF] are reflected in this model, with overexpression of AhR and its canonical targets COX-2 and C/EBPβ along with down-regulation of AhRR. Notably, we demonstrate that AhRR overexpression in the PyMT background increases tumor latency and decreases tumor incidence and burden. While lung metastases are prevalent in PyMT/wt mice, as expected, we found lung metastases in only 40% of PyMT/AhRR + mice at necropsy. This may be an outcome of decreased primary tumor burden in PyMT/AhRR + mice and/or may reflect a decrease in functional metastatic capacity in PyMT/AhRR + tumor cells. Previous studies have found that AhRR silencing increases tumor cell migration and invasion [START_REF] Zudaire | The aryl hydrocarbon receptor repressor is a putative tumor suppressor gene in multiple human cancers[END_REF]. Further, breast cancer patients who retain high AhRR expression show prolonged metastasis-free survival [START_REF] Vacher | High AHR expression in breast tumors correlates with expression of genes from several signaling pathways namely inflammation and endogenous tryptophan metabolism[END_REF], strongly suggesting that AhRR plays a functional role in limiting biological behaviors which contribute to metastasis. In earlier studies, we demonstrated that activation of C/EBPβ drives AhR-mediated COX-2 gene induction via activation of PKA [START_REF] Vogel | Regulation of prostaglandin endoperoxide H synthase-2 induction by dioxin in rat hepatocytes: possible c-Src-mediated pathway[END_REF][START_REF] Vogel | Dioxin increases C/EBPbeta transcription by activating cAMP/protein kinase A[END_REF]. Therefore, it is not unlikely that intrinsic as well as hostdependent effects of AhRR are mediated through repression of the PKA/C/EBPβ pathway causing inhibition of tumor growth. PKA has been found to control cell growth in many cancer types in vivo and in vitro and represents a potential target for pharmacological treatment of tumors [START_REF] Sapio | Targeting protein kinase A in cancer therapy: an update[END_REF]. Downstream of PKA, phosphorylation of Src has been shown to initiate mammary cell transformation associated with increased cell proliferation [START_REF] Beristain | PKA signaling drives mammary tumorigenesis through Src[END_REF]. High Src expression has also been defined with basal-like and HER2 human breast cancer associated with poor clinical outcome [START_REF] Elsberger | Is expression or activation of Src kinase associated with cancer-specific survival in ER-, PR-and HER2-negative breast cancer patients?[END_REF]. Notably, AhR-regulated induction of COX-2 is mediated through a mechanism involving activation of PKA and Src signaling downstream [START_REF] Vogel | Regulation of prostaglandin endoperoxide H synthase-2 induction by dioxin in rat hepatocytes: possible c-Src-mediated pathway[END_REF][START_REF] Vogel | Dioxin increases C/EBPbeta transcription by activating cAMP/protein kinase A[END_REF].

Interestingly, the selective inhibition of COX-2 has been shown to significantly increase apoptosis in tumors and to decrease the number and size of tumors in the PyMT mouse model [START_REF] Buchanan | Targeting cyclooxygenase-2 and the epidermal growth factor receptor for the prevention and treatment of intestinal cancer[END_REF][START_REF] Esbona | COX-2 modulates mammary tumor progression in response to collagen density[END_REF] Resistance to chemotherapy is a major factor driving breast cancer mortality, particularly in TNBC where targeted therapies are not available for most patients. We report here that AhRR overexpression sensitizes PyMT-derived mammary tumor cells and human breast cancer cells to both Dox and EtOP.

AhRR overexpression is as effective as AhR deletion, suggesting that AhRR restoration is a feasible approach for addressing chemoresistance. The elevated levels of γH2AX observed in AhRcompromised breast cancer cells exposed to genotoxic drugs, supports previous studies showing that AhR plays an important role in repair of DSB [START_REF] Chan | TCDD affects DNA double strand-break repair[END_REF][START_REF] Dittmann | The nuclear aryl hydrocarbon receptor is involved in regulation of DNA repair and cell survival following treatment with ionizing radiation[END_REF][START_REF] Pollet | The AHR represses nucleotide excision repair and apoptosis and contributes to UV-induced skin carcinogenesis[END_REF]. In several malignancies, including a proportion of triple negative breast cancers, elevated DSB repair activities impair therapeutic efficacy by enhancing the resistance towards therapeutically induced DNA damage [START_REF] Gasparini | Protective[END_REF]67). Interestingly, C/EBPβ was shown to protect ovarian cancers against cisplatin treatment by enforcing the expression of genes involved in drug transport, cell survival, and DNA repair, more precisely homologous recombination repair and non-homologous end-joining [START_REF] Liu | C/EBPbeta enhances platinum resistance of ovarian cancer cells by reprogramming H3K79 methylation[END_REF]. However, to what extent AhR's impact on DSB repair and apoptosis depends on C/EBPβ is not well understood and currently under investigation.

In summary our results demonstrate that AhRR overexpression suppresses mammary tumor growth and progression and is associated with the repression of markers of inflammation and tumor cell survival, particularly if the AhR is constantly activated by endogenous or persistent toxic environmental ligands (Fig. 8). It is important to note that AhR signaling in mammary microenvironment cells has been observed previously. For example, growth of mouse mammary fibroblasts as leimyosarcomas in vivo was decreased by AhR deletion [START_REF] Mulero-Navarro | Immortalized mouse mammary fibroblasts lacking dioxin receptor have impaired tumorigenicity in a subcutaneous mouse xenograft model[END_REF]. In addition, human breast cancerassociated fibroblasts upregulate the canonical AhR target gene, CYP1B1, and proliferate in response to treatment with the AhR ligand, 3-methylcholanthrene (68). While our results suggest that AhRR overexpression in the host environment is sufficient to decrease mammary tumor cell growth, the tumor cell-intrinsic vs. extrinsic roles of AhR/AhRR have to be explored in more detail. Additional studies are needed and will address approaches to functionally restore AhRR in AhRR silenced tumors.

Author

  AhRR or A-C/EBP transfected UCD-PYMT cells were seeded at a density of 2 × 10 4 per mL of growth medium in 48-well plates and were incubated overnight. At 24, 48, 72, and 96 h, 20 μL (5 g/L) of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide)

3. 2 .

 2 AhRR increases tumor latency and decreases tumor incidence in the PyMT model. To further define the role of AhRR in mammary tumorigenesis, we chose the polyoma middle T antigen (PyMT) model, a widely used model of metastatic breast cancer. As shown in Figure 2, the PyMT model reflects expression changes observed in human breast cancer. Specifically, expression of AhR increases in mammary tissue during tumor progression with expression of AhR gene targets and inflammatory markers (e.g. COX-2 and C/EBPβ) increasing accordingly. In contrast, the expression of AhRR decreases, suggesting that the healthy "yin and yang" of AhR and AhRR is disrupted, favoring AhR signaling.

  AhRR overexpressing cells, for both Dox and EtOP. AhRR overexpression augmented apoptosis in response to both Dox and EtOP and mitigated the rescue provided by TCDD. This suggests that functional restoration of AhRR to breast cancer cells may be useful in addressing chemoresistance, a major driver of breast cancer mortality. Next, we investigated whether a modulation of AhR activity affects drug-induced apoptosis consistently in human triple negative MDA-MB 231 breast cancer cells (Fig.7A). Moreover, experiments with luminal ER-positive MCF-7 cells were included (Fig.7C) to explore if AhRR also mediates apoptosis in non TNBC breast cancer cells. Whereas MDA-MB 231 cells were treated for 48 h with the genotoxic drugs, MCF-7 cells were incubated for 72 h. The efficacy of the drugs largely depends on the proliferation rate of the cells.Given that the doubling time of MCF-7 cells (approx. 43 h) is significantly longer than the doubling time of MDA-MB 231 cells (approx. 31 h) as reported[START_REF] Sweeney | Lack of relationship between CDK activity and G1 cyclin expression in breast cancer cells[END_REF] we treated the MCF-7 cells 24 h longer than the MDA-MB 231 cells, resulting roughly in the same number of cell divisions before the measurements. Hence, the difference in treatment time may explain that both AhRR-transfected celllines exhibit a comparable pro-apoptotic effect. Transient overexpression of rat AhRR or CRISPR/Cas9-mediated AhR knockout resulted in a similarly enhanced susceptibility of both cell lines towards Dox-and EtOP-induced apoptosis (Fig.7A and C). Ectopic overexpression of the rat AhRR has been found to effectively antagonize AhR in human HepG2 and HaCaT cells[START_REF] Haarmann-Stemmann | Regulation of dioxin receptor function by different beta-carboline alkaloids[END_REF][START_REF] Pollet | The AHR represses nucleotide excision repair and apoptosis and contributes to UV-induced skin carcinogenesis[END_REF].Overexpression of AhRR in AhR knockout cells yielded a similar level of apoptosis as either condition alone. Collectively, these data suggest that the pro-apoptotic effect of AhRR largely depends on AhR inhibition. As Dox and EtOP induce DNA double-strand breaks (DSB) to initiate apoptosis, we next analyzed the number of cells positive for phosphorylated histone 2AX (γH2AX), an established marker

Further, we tested

  whether AhRR affects the expression of COX-2 and C/EBPβ as well as the growth of tumor cells in vitro using UCD-PYMT, a tumor cell line isolated from a PyMT mammary tumor. Activation of AhR by TCDD of these cells led to an increase in both COX-2 and C/EBPβ, with significant inhibition of this response by AhRR overexpression. Moreover, the constitutive and TCDD-stimulated AhR-mediated DRE reporter activity as well as C/EBPβ DNA binding was abrogated by AhRR overexpression in UCD-PYMT cells, which was associated with the inhibition of cell proliferation. The results are in line with the in vivo findings and confirm previous reports with human breast cancer cellsshowing a reduced cell proliferation and increased apoptosis by AhRR overexpression[START_REF] Zudaire | The aryl hydrocarbon receptor repressor is a putative tumor suppressor gene in multiple human cancers[END_REF][START_REF] Wong | Characterization of MCF mammary epithelial cells overexpressing the Aryl hydrocarbon receptor (AhR)[END_REF][START_REF] Kanno | The inhibitory effect of aryl hydrocarbon receptor repressor (AhRR) on the growth of human breast cancer MCF-7 cells[END_REF]. Our previous studies found that C/EBPβ inducing interleukin 1β (IL-1β) and COX-2 are important mediators of an AhR-dependent and TCDD-induced resistance to apoptosis in lymphoma cells, demonstrating their critical role in AhR-driven tumor cell survival[START_REF] Vogel | A Protective Role of Aryl Hydrocarbon Receptor Repressor in Inflammation and Tumor Growth[END_REF][START_REF] Vogel | Pathogenesis of aryl hydrocarbon receptor-mediated development of lymphoma is associated with increased cyclooxygenase-2 expression[END_REF]. In addition to a host-dependent tumor suppressive effect of AhRR indicated by data from the mouse xenograft model, the results with mammary tumor cells suggest that AhRR mediates also cell-intrinsic responses possibly through the suppression of C/EBPβ and COX-2. COX-2 is an inducible isoform upregulated in many cancers[START_REF] Wang | Eicosanoids and cancer[END_REF].

Figure legends Figure 1 .

 legends1 Figure legends

Figure 2 .Figure 3 .

 23 Figure 2. mRNA Expression of marker genes (A) AhR, (B) AhRR, (C) COX-2 and (D) C/EBPβ in mammary tissue during tumor development from 4 to 12 weeks in PyMT mice (n=15) compared to wt mice (n=15). NS=not significant; plot of single data points of 15 mice in each group and t-test statistics are shown. Significantly different **p < 0.01, ***p < 0.001, ****p < 0.0001

Figure 4 .Fig. 5 .Fig. 6 .

 456 Figure 4. Whole-mounted mammary glands and lungs from PyMT/wt and PyMT/AhRR + mice. Virgin mammary glands from 5-month-old (A) PyMT/wt and (B) PyMT/AhRR + transgenic female mice. Whole-mounts were spread on slides, fixed and stained with hematoxylin in order to elucidate ductal structure. Mammary glands from 5-month old PyMT/wt mice compared with a tumor free PyMT/AhRR + transgenic female mice are shown. Arrows indicate MT (mammary tumors) and intramammary LN (lymph node). Overexpression of AhRR in PyMT mice suppressed lung metastasis in PyMT mice. Whole-mounted lungs from (C) PyMT/wt and (D) PyMT/AhRR + mice at 5 months after birth. Whole mounts were spread on slides, fixed and stained with hematoxylin in order to elucidate metastatic lung tumors (Mets) as indicated. (E) Percentage of ten PyMT/wt and PyMT/AhRR + mice with lung metastasis at necropsy. Fisher's exact test was applied, * Statistically significant different, P = 0.0108

Fig. 7 .Fig. 8 AFigure 1 Fig. 5 . 2 mFigure 3 Figure 5

 7815235 Fig. 7. AhR deficiency and AhRR overexpression enhance drug-induced apoptosis and doublestrand breaks (DSB) in human MDA-MB 231 and MCF-7 cells. Transiently AhRR overexpressing, AhRknockout and control (A, B) MDA-MB 231 and (C, D) MCF-7 cells were treated with Dox and EtOP as indicated. After 48 h (MDA-MB 231) and 72 h (MCF-7), (A, C) apoptosis (percentage of cells in subG1) and (B, D) number of γH2AX-positive indicating DSB breast cancer cells were determined. Statistical significance was tested with a two-way ANOVA and multiplicity adjusted P values were computed with Tukey's post hoc test. *, p < 0.05 compared to the respective DMSO treated sample. #, p < 0.05 compared to the respective wt/EV sample. (E) mRNA expression of AhR and (F) protein level of AhR in MDA-MB 231 and MCF-7 wt and AhR knockout cells.

Figure 6 2 PKA

 62 Figure 6 A and B

  EBPβ have been associated with the progression of breast and ovarian cancers and are correlated with an unfavorable prognosis[START_REF] Liu | C/EBPbeta enhances platinum resistance of ovarian cancer cells by reprogramming H3K79 methylation[END_REF][START_REF] Milde-Langosch | Expression of the CCAAT/enhancer-binding proteins C/EBPalpha, C/EBPbeta and C/EBPdelta in breast cancer: correlations with clinicopathologic parameters and cell-cycle regulatory proteins[END_REF][START_REF] Kurzejamska | C/EBPbeta expression is an independent predictor of overall survival in breast cancer patients by MHCII/CD4-dependent mechanism of metastasis formation[END_REF]. This is supported by studies on different mouse models for metastatic breast cancer, showing that C/EBPβ induces the expression of genes relevant for metastasis to the lungs[START_REF] Kurzejamska | C/EBPbeta expression is an independent predictor of overall survival in breast cancer patients by MHCII/CD4-dependent mechanism of metastasis formation[END_REF][START_REF] Wiegmans | Rad51 supports triple negative breast cancer metastasis[END_REF]. Interestingly, Wiegmans et al. reported that C/EBPβ cooperates

	with RAD51, a key protein of homologous recombination repair, to control invasion-and metastasis-
	associated gene expression (62).

. Degner et al. (58) have shown that AhR ligands can upregulate COX-2 expression, which led to a proinflammatory local environment that supported tumor development. Furthermore, elevated expression levels of C/

Acknowledgments: We like to thank Jane Chen, Center for Comparative Medicine, University of California, Davis, for technical assistance and Charles Vinson for providing critical plasmid constructs.

Funding: Research reported in this publication is supported in part by the National Institute of Environmental Health Sciences of the National Institutes of Health under Award Number R01 ES029126 and R21ES030419, the core center grant, P30-ES023513 from the National Institute of Environmental Health Sciences, and by the University of California Davis Cancer Center support grant P30 CA093373 and the Cancer Immunology Grant fund 48649. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health. Research in the lab of T.H.S. is supported by the Deutsche Forschungsgemeinschaft (HA 7346/2-2).

Conflicts of Interest:

The authors declare no conflict of interest.