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,

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.

Prothrombinase activates prothrombin through initial cleavage at Arg320 followed by cleavage at Arg271. This pathway is characterized by the generation of an enzymatically active, transient intermediate, meizothrombin, that has increased chromogenic substrate activity but poor clotting activity. The heavy chain of factor Va contains an acidic region at the COOH terminus (residues 680−709). We have shown that a pentapeptide from this region (DYDYQ) inhibits prothrombin activation by prothrombinase by inhibiting meizothrombin generation. To ascertain the function of these regions, we have created a mutant recombinant factor V molecule that is missing the last 30 amino acids from the heavy chain (factor VΔ680−709) and a mutant molecule with the 695DYDY698 → AAAA substitutions (factor V4A). The clotting activities of both recombinant mutant factor Va molecules were impaired compared to the clotting activity of wild-type factor Va (factor VaWt). Using an assay employing purified reagents, we found that prothrombinase assembled with factor VaΔ680−709 displayed an ∼39% increase in kcat, while prothrombinase assembled with factor Va4A exhibited an ∼20% increase in kcat for the activation of prothrombin as compared to prothrombinase assembled with factor VaWt. Gel electrophoresis analyzing prothrombin activation by prothrombinase assembled with the mutant molecules revealed a delay in prothrombin activation with persistence of meizothrombin. Our data demonstrate that the COOH-terminal region of factor Va heavy chain is indeed crucial for coordinated prothrombin activation by prothrombinase because it regulates meizothrombin cleavage at Arg271 and suggest that this portion of factor Va is partially responsible for the enhanced procoagulant function of prothrombinase.

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We have demonstrated that amino acids E323, Y324, E330, and V331 from the factor Va heavy chain are required for the interaction of the cofactor with factor Xa and optimum rates of prothrombin cleavage. We have also shown that amino acid region 332−336 contains residues that are important for cofactor function. Using overlapping peptides, we identified amino acids D334 and Y335 as contributors to cofactor activity. We constructed recombinant factor V molecules with the mutations D334 → K and Y335 → F (factor VKF) and D334 → A and Y335 → A (factor VAA). Kinetic studies showed that while factor VaKF and factor VaAA had a KD for factor Xa similar to the KD observed for wild-type factor Va (factor VaWT), the clotting activities of the mutant molecules were impaired and the kcat of prothrombinase assembled with factor VaKF and factor VaAA was reduced. The second-order rate constant of prothrombinase assembled with factor VaKF or factor VaAA for prothrombin activation was ∼10-fold lower than the second-order rate constant for the same reaction catalyzed by prothrombinase assembled with factor VaWT. We also created quadruple mutants combining mutations in the amino acid region 334–335 with mutations at the previously identified amino acids that are important for factor Xa binding (i.e., E323Y324 and E330V331). Prothrombinase assembled with the quadruple mutant molecules displayed a second-order rate constant up to 400-fold lower than the values obtained with prothrombinase assembled with factor VaWT. The data demonstrate that amino acid region 334–335 is required for the rearrangement of enzyme and substrate necessary for efficient catalysis of prothrombin by prothrombinase.

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.

Background Activated protein C (APC) inactivates membrane-bound factor Va following cleavages of the heavy chain at Arg306, Arg506, and Arg679. Objectives To examine which cleavage is most important for inactivation, recombinant factor V molecules were constructed as follows: factor V306Q (R306→Q), factor V506Q (R506→Q), and factor V306Q/506Q (R306→Q and R506→Q). Methods The recombinant molecules were expressed in mammalian cells, purified, and assayed prior and after incubation with APC and lipids for 30 min (factor Vai) in clotting assays and in an assay using purified reagents and saturating concentrations of factor Va. Results Clotting assays demonstrated that factor VaiWT, factor Vai306Q and factor Vai506Q were devoid of activity, while factor Vai306Q/506Q maintained ~70% activity following a 30 minute incubation with APC. Prothrombinase assembled with all mutant cofactor molecules before and after treatment with APC had Km values similar to values found with prothrombinase assembled with factor VaWT. Prothrombinase assembled with factor VaiWT demonstrated 20-fold reduction in kcat, while prothrombinase assembled with factor Vai506Q had a 2-fold reduction in kcat as compared to prothrombinase assembled with factor VaWT. In contrast, factor Vai306Q and factor Vai306Q/506Q didn’t show any loss in kcat under similar experimental conditions. Conclusion Our data demonstrate that: 1) the activity of an APC-treated factor Va molecule bearing a single mutation at Arg306 or Arg506 depends on the assay used; and 2) regardless of the assay employed, in the absence of the APC-cleavage sites at Arg306 and Arg506, the active cofactor is unable to be significantly inactivated by APC in the presence of a membrane surface.

The prothrombinase complex catalyzes the activation of prothrombin to α-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 and 695−698 as follows: factor V3K (334DY335 → KF and 695DYDY698 → KFKF), factor VKF/4A (334DY335 → KF and 695DYDY698 → AAAA), and factor V6A (334DY335 → AA and 695DYDY698 → AAAA). The recombinant factor V molecules were expressed and purified to homogeneity. Factor Va3K, factor VaK4/4A, and factor Va6A had reduced affinity for factor Xa, when compared to the affinity of the wild-type molecule (factor VaWt) for the enzyme. Prothrombinase assembled with saturating concentrations of factor Va3K had a 6-fold reduced second-order rate constant for prothrombin activation compared to the value obtained with prothrombinase assembled with factor VaWt, while prothrombinase assembled with saturating concentrations of factor VaKF/4A and factor Va6A had approximately 1.5-fold reduced second-order rate constants. Overall, the data demonstrate that amino acid region 334–335 together with amino acid region 695−698 from factor Va heavy chain are part of a cooperative mechanism within prothrombinase regulating cleavage and activation of prothrombin by factor Xa.