Aryl hydrocarbon receptor regulates cell cycle progression in human breast cancer cells via a functional interaction with cyclin-dependent kinase 4

ABSTRACT The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor with constitutive activities and those induced by xenobiotic ligands, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). One unexplained cellular role for the AHR is its ability to promote cell cycle progression in the absence of exogenous ligands, whereas treatment with exogenous ligands induces cell cycle arrest. Within the cell cycle, progression from G 1 to S phase is controlled by sequential phosphorylation of the retinoblastoma protein (RB1) by cyclin D-cyclin-dependent kinase (CDK) 4/6 complexes. In this study, the functional interactions between the AHR, CDK4, and cyclin D1 (CCND1) were investigated as a potential mechanism for the cell cycle regulation by the AHR. Time course cell cycle and molecular experiments were performed in human breast cancer cells. The results demonstrated that the AHR and CDK4 interact within the cell cycle, and the interaction was disrupted upon TCDD treatment. The disruption was temporally correlated with G 1 cell cycle arrest and decreased phosphorylation of RB1. Biochemical reconstitution assays using in vitro-translated protein recapitulated the AHR and CDK4 interaction and showed that CCND1 was also part of the complex. In vitro assays for CDK4 kinase activity demonstrated that RB1 phosphorylation by the AHR/CDK4/CCND1 complex was reduced in the presence of TCDD. The results suggest that the AHR interacts in a complex with CDK4 and CCND1 in the absence of exogenous ligands to facilitate cell cycle progression. This interaction is disrupted by exogenous ligands, such as TCDD, to induce G 1 cell cycle arrest. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor and a member of the basic helix-loophelix, period/aryl hydrocarbon receptor nuclear translocator (ARNT)/single-minded (PAS) superfamily. In the canonical model for AHR signaling, the unliganded form of the receptor exists in the cytoplasm in a stable complex with HSP90

Cooperative Regulation of the Activity of Factor Xa within Prothrombinase by Discrete Amino Acid Regions from Factor Va Heavy Chain†

ABSTRACT: The prothrombinase complex catalyzes the activation of prothrombin to R-thrombin. We have repetitively shown that amino acid region 695DYDY698 from the COOH terminus of the heavy chain of factor Va regulates the rate of cleavage of prothrombin at Arg271 by prothrombinase. We have also recently demonstrated that amino acid region 334DY335 is required for the optimal activity of prothrombinase. To assess the effect of these six amino acid residues on cofactor activity, we created recombinant factor Va molecules combining mutations at amino acid regions 334–335 an

Breast Cancer Stem-Like Cells Are Inhibited by a Non-Toxic Aryl Hydrocarbon Receptor Agonist

Cancer stem cells (CSCs) have increased resistance to cancer chemotherapy. They can be enriched as drug-surviving CSCs (D-CSCs) by growth with chemotherapeutic drugs, and/or by sorting of cells expressing CSC markers such as aldehyde dehydrogenase-1 (ALDH). CSCs form colonies in agar, mammospheres in low-adherence cultures, and tumors following xenotransplantation in Scid mice. We hypothesized that tranilast, a non-toxic orally active drug with anti-cancer activities, would inhibit breast CSCs.We examined breast cancer cell lines or D-CSCs generated by growth of these cells with mitoxantrone. Tranilast inhibited colony formation, mammosphere formation and stem cell marker expression. Mitoxantrone-selected cells were enriched for CSCs expressing stem cell markers ALDH, c-kit, Oct-4, and ABCG2, and efficient at forming mammospheres. Tranilast markedly inhibited mammosphere formation by D-CSCs and dissociated formed mammospheres, at pharmacologically relevant concentrations. It was effective against D-CSCs of both HER-2+ and triple-negative cell lines. Tranilast was also effective in vivo, since it prevented lung metastasis in mice injected i.v. with triple-negative (MDA-MB-231) mitoxantrone-selected cells. The molecular targets of tranilast in cancer have been unknown, but here we demonstrate it is an aryl hydrocarbon receptor (AHR) agonist and this plays a key role. AHR is a transcription factor activated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polycyclic aromatic hydrocarbons and other ligands. Tranilast induced translocation of the AHR to the nucleus and stimulated CYP1A1 expression (a marker of AHR activation). It inhibited binding of the AHR to CDK4, which has been linked to cell-cycle arrest. D-CSCs expressed higher levels of the AHR than other cells. Knockdown of the AHR with siRNA, or blockade with an AHR antagonist, entirely abrogated the anti-proliferative and anti-mammosphere activity of tranilast. Thus, the anti-cancer effects of tranilast are AHR dependent.We show that tranilast is an AHR agonist with inhibitory effects on breast CSCs. It is effective against CSCs of triple-negative breast cancer cells selected for anti-cancer drug resistance. These results suggest it might find applications in the treatment of breast cancer

Activation of the Aryl-Hydrocarbon Receptor Inhibits Invasive and Metastatic Features of Human Breast Cancer Cells and Promotes Breast Cancer Cell Differentiation

The current statistics associated with breast cancer continue to show a relatively high recurrence rate together with a poor survival for aggressive metastatic disease. These findings reflect, in part, the pharmaceutical intractability of processes involved in the metastatic process and highlight the need to identify additional drug targets for the treatment of late-stage disease. In the current study, we report that ligand activation of the aryl-hydrocarbon receptor (AhR) inhibits multiple aspects of the metastatic process in a panel of breast cancer cell lines that represent the major breast cancer subtypes. Specifically, it was observed that treatment with exogenous AhR agonists significantly inhibited cell invasiveness and motility in the Boyden chamber assay and inhibited colony formation in soft agar regardless of estrogen receptor (ER), progesterone receptor, or human epidermal growth factor receptor 2 status. Knockdown of the AhR using small interfering RNA duplexes demonstrated that the inhibition of invasiveness was receptor dependent and that endogenous receptor activity was protective in each cell type examined. The inhibition of invasiveness and anchorage-independent growth correlated with the ability of exogenous AhR agonists to promote differentiation. Finally, exogenous AhR agonists were able to promote differentiation in a putative mammary cancer stem cell line. Cumulatively, these results suggest that the AhR plays an important role in mammary epithelial differentiation and, as such, represent a promising therapeutic target for a range of phenotypically distinct human breast cancers