Towards Resolving the Pro- and Anti-Tumor Effects of the Aryl Hydrocarbon Receptor.

We have postulated that the aryl hydrocarbon receptor (AHR) drives the later, more lethal stages of some cancers when chronically activated by endogenous ligands. However, other studies have suggested that, under some circumstances, the AHR can oppose tumor aggression. Resolving this apparent contradiction is critical to the design of AHR-targeted cancer therapeutics. Molecular (siRNA, shRNA, AHR repressor, CRISPR-Cas9) and pharmacological (AHR inhibitors) approaches were used to confirm the hypothesis that AHR inhibition reduces human cancer cell invasion (irregular colony growth in 3D Matrigel cultures and Boyden chambers), migration (scratch wound assay) and metastasis (human cancer cell xenografts in zebrafish). Furthermore, these assays were used for a head-to-head comparison between AHR antagonists and agonists. AHR inhibition or knockdown/knockout consistently reduced human ER−/PR−/Her2− and inflammatory breast cancer cell invasion, migration, and metastasis. This was associated with a decrease in invasion-associated genes (e.g., Fibronectin, VCAM1, Thrombospondin, MMP1) and an increase in CDH1/E-cadherin, previously associated with decreased tumor aggression. Paradoxically, AHR agonists (2,3,7,8-tetrachlorodibenzo-p-dioxin and/or 3,3′-diindolylmethane) similarly inhibited irregular colony formation in Matrigel and blocked metastasis in vivo but accelerated migration. These data demonstrate the complexity of modulating AHR activity in cancer while suggesting that AHR inhibitors, and, under some circumstances, AHR agonists, may be useful as cancer therapeutics.

An Aryl Hydrocarbon Receptor-Mediated Amplification Loop That Enforces Cell Migration in ER-/PR-/Her2- Human Breast Cancer Cells.

The endogenous ligand-activated aryl hydrocarbon receptor (AHR) plays an important role in numerous biologic processes. As the known number of AHR-mediated processes grows, so too does the importance of determining what endogenous AHR ligands are produced, how their production is regulated, and what biologic consequences ensue. Consequently, our studies were designed primarily to determine whether ER-/PR-/Her2- breast cancer cells have the potential to produce endogenous AHR ligands and, if so, how production of these ligands is controlled. We postulated that: 1) malignant cells produce tryptophan-derived AHR ligand(s) through the kynurenine pathway; 2) these metabolites have the potential to drive AHR-dependent breast cancer migration; 3) the AHR controls expression of a rate-limiting kynurenine pathway enzyme(s) in a closed amplification loop; and 4) environmental AHR ligands mimic the effects of endogenous ligands. Data presented in this work indicate that primary human breast cancers, and their metastases, express high levels of AHR and tryptophan-2,3-dioxygenase (TDO); representative ER-/PR-/Her2- cell lines express TDO and produce sufficient intracellular kynurenine and xanthurenic acid concentrations to chronically activate the AHR. TDO overexpression, or excess kynurenine or xanthurenic acid, accelerates migration in an AHR-dependent fashion. Environmental AHR ligands 2,3,7,8-tetrachlorodibenzo[p]dioxin and benzo[a]pyrene mimic this effect. AHR knockdown or inhibition significantly reduces TDO2 expression. These studies identify, for the first time, a positive amplification loop in which AHR-dependent TDO2 expression contributes to endogenous AHR ligand production. The net biologic effect of AHR activation by endogenous ligands, which can be mimicked by environmental ligands, is an increase in tumor cell migration, a measure of tumor aggressiveness.

In silico identification of an aryl hydrocarbon receptor antagonist with biological activity in vitro and in vivo.

The aryl hydrocarbon receptor (AHR) is critically involved in several physiologic processes, including cancer progression and multiple immune system activities. We, and others, have hypothesized that AHR modulators represent an important new class of targeted therapeutics. Here, ligand shape-based virtual modeling techniques were used to identify novel AHR ligands on the basis of previously identified chemotypes. Four structurally unique compounds were identified. One lead compound, 2-((2-(5-bromofuran-2-yl)-4-oxo-4H-chromen-3-yl)oxy)acetamide (CB7993113), was further tested for its ability to block three AHR-dependent biologic activities: triple-negative breast cancer cell invasion or migration in vitro and AHR ligand-induced bone marrow toxicity in vivo. CB7993113 directly bound both murine and human AHR and inhibited polycyclic aromatic hydrocarbon (PAH)- and TCDD-induced reporter activity by 75% and 90% respectively. A novel homology model, comprehensive agonist and inhibitor titration experiments, and AHR localization studies were consistent with competitive antagonism and blockade of nuclear translocation as the primary mechanism of action. CB7993113 (IC50 3.3 × 10(-7) M) effectively reduced invasion of human breast cancer cells in three-dimensional cultures and blocked tumor cell migration in two-dimensional cultures without significantly affecting cell viability or proliferation. Finally, CB7993113 effectively inhibited the bone marrow ablative effects of 7,12-dimethylbenz[a]anthracene in vivo, demonstrating drug absorption and tissue distribution leading to pharmacological efficacy. These experiments suggest that AHR antagonists such as CB7993113 may represent a new class of targeted therapeutics for immunomodulation and/or cancer therapy.

Immunoglobulin light chain, Blimp-1 and cytochrome P4501B1 peptides as potential vaccines for AL amyloidosis.

Amyloid light chain (AL) amyloidosis is a lethal disorder characterized by the pathologic deposition of clonal plasma cell-derived, fibrillogenic immunoglobulin light chains in vital organs. Current chemotherapeutic regimens are problematic in patients with compromised organ function and are not effective for all patients. Here, a platform of computer-based prediction and preclinical mouse modeling was used to begin development of a complementary, immunotherapeutic approach for AL amyloidosis. Three peptide/MHC I-binding algorithms identified immunogenic peptides from three AL plasma cell-associated proteins: (1) amyloidogenic λ6 light chains, (2) CYP1B1, a universal tumor antigen hyper-expressed in AL plasma cells and (3) B lymphocyte-induced maturation protein 1 (Blimp-1), a transcription factor required for plasma cell differentiation. The algorithms correctly predicted HLA-A(*)0201-binding native and heteroclitic peptides. In HLA-A2 transgenic mice, these peptides, given individually or in combination, induced potent CTL which kill peptide-loaded human lymphoma cells and/or lymphoma cells producing target protein. Blimp-1 peptide-immunized mice exhibited a reduced percentage of splenic, lymph node and bone marrow plasma cells and a decrease in the absolute number of splenic plasma cells demonstrating (1) presentation of target peptide by endogenous plasma cells and (2) appropriate CTL homing to lymphoid organs followed by killing of target plasma cells. These studies suggest that AL amyloidosis, with its relatively low tumor cell burden, may be an attractive target for peptide-based multivalent vaccines.