Constitutively-active Receptor Research Articles

Abstract A049: The sfRon kinase as a potential novel target for breast cancer therapy

Abstract Background: Our recent data revealed striking predominance of an alternatively transcribed form of the Ron receptor tyrosine kinase, known as short-form Ron (sfRon), in breast cancer. sfRon is the major active Ron isoform in breast tumors from patients, and has remarkable effects on metastasis. sfRon lacks the extracellular ligand-binding domain, but organizes into a constitutively-active receptor. Our in vitro studies revealed that overexpression of sfRon in breast cancer cell lines promotes aggressive phenotypes including an epithelial-mesenchymal transition and invasion through basement membranes. We observed that sfRon strongly signals through the PI3K pathway and that interaction between sfRon and PI3K was required for sfRon function. In vivo studies revealed striking results that expression of sfRon was sufficient to convert slow growing non-metastatic tumors into fast growing tumors that spontaneously metastasized from the mammary gland to liver, spleen and bones. An increased understanding of how sfRon contributes to tumor progression and metastasis is critical. Such data carries additional importance because sfRon does not require ligand binding for activity, appears to function in a manner distinct from full length Ron, and may not be targetable using standard approaches such as antibody inhibitors (e.g. RON8, which is now being tested in clinical trials). Instead, our data suggest that inhibition of Ron kinase activity, which would block both Ron and sfRon, would be preferable and that inhibition of downstream PI3K might also be effective in breast tumors expressing sfRon. Results: Through collaborations with various pharmaceutical companies we have obtained several compounds that specifically target Ron/sfRon kinase (e.g. ASLAN002) or PI3 kinase (e.g. BKM120). Our in vitro data demonstrate that ASLAN002 inhibits the activity of sfRon kinase in MCF7 breast cancer cells. The drug also completely blocked PI3K signaling downstream of sfRon. Our studies also showed BKM120 to be a potent inhibitor of PI3K pathway in sfRon-expressing breast cancer cells, effective in doses as low as 250 nM. We have also shown that, while the RON8 antibody inhibits activity of full length Ron kinase, it is not effective against sfRon activity. Based on these promising in vitro results, we extended our work to in vivo assays. We used NOD/SCID mice to orthotopically transplant sfRon-expressing MCF7 cells. After tumors reached 200mm3 we initiated treatment every other day with vehicle control, RON8 antibody, ASLAN002, BKM120, or ASLAN002+BKM120 combination therapy. Our data showed that although the RON8 antibody designed to target Ron is not effective against tumors expressing sfRon, monotherapy with ASLAN002 or BKM120 significantly inhibited tumor growth. However, the most effective treatment regimen was ASLAN002+BKM120 combination therapy. Most importantly, we also observed reduced formation of metastasis in mice treated with ASLAN002, BKM120, or the combination therapy compared to vehicle controls (% of spontaneous metastasis: 44%, 44%, and 33% vs 75%, respectively). Conclusion: Our experimental approach supports the novel concept to use Ron and PI3K pathway inhibitors as potential new therapeutic strategies in breast cancer. These data could have significant impact on future clinical trials for both Ron and PI3K inhibitors, which has high potential to impact patients in the short-term. Citation Format: Magdalena Bieniasz, Najme Faham, Alana L. Welm. The sfRon kinase as a potential novel target for breast cancer therapy. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A049.

(Far) Outside the box: Genomic approach to acute porphyria

If I were living in Caucasus I would be writing fairy tales there Chekov, 1888 The question of the reasons for the extreme variation in morbidity among the gene carriers of acute porphyria and the great diversity of the precipitating factors are approached by the aid of a model of interacting genomic circuits. It is based on the current paradigm of the acute porphyric attack as a result of a toxic proximal overload of the enzyme-deficient heme-biosynthetic patway. Porphyrogenic influx of precursors is seen as a consequence of uncontrolled induction of its gate-keeping enzyme, ubiquitous 5-aminolevulinate synthase (ALAS1), due to attenuated post-translational control of the enzyme combined with activated gene transcription. Focus is directed on the genomic control of the master-regulator of ALAS1-transcription, the nuclear receptor pair constitutively active receptor (CAR) and pregnane xenobiotic receptor (PXR). On activation by their ligands, i.e. lipophilic drugs, solvents, alcohols, hormonal steroids and biocides, these DNA-binding proteins transform xenobiotic or steroid stimuli to coordinated activations of gene transcription-programs for ALAS1 and apo-cytochromes P450 (apo-CYPs), thus effecting the formation of xenobiotic-metabolizing cytochrome P450 enzymes. The potency of the CAR/PXR-transduction axis is enhanced by co-activators generated in at least four other genomic circuits, each triggered by different external and internal stimuli clinically experienced to be porphyrogenic, and each controlled by co-activating and co-repressing modulators. The expressions of the genes for CAR and PXR are thus augmented by binding glucocorticoid receptor (GR) activated by a steroid hormone, e.g, cortisol generated in fasting, infection or different forms of stress. The promotor regions of ALAS1 and apoCYPs contain binding sites for at least three co-activating transcription factors enhancing CAR/PXR transduction: i.e. the ligand-independent growth hormone (GH)-pulse controlled hepatocyte nuclear factor 4 (HNF4), the insulin-responsive forkhead box class O-(FOXO) protein pathway activated in stress and infection, and the proliferator-activated receptor gamma co-activator 1 alpha (PGC-1alpha) circuit responding to glucagon liberated in fasting. Many interactions and cross-talk take place within the tangle of genomic circuits that control ALAS1-transcription, which may explain the extreme inter- and intra-individual variability in morbidity in acute porphyria. Reasons for gender-differences are found in sex-dependent control of HPA- and GH-activity as well as in direct, or GR-mediated effects on CAR/PCR activation. Constitutional differences in individual porphyric morbidity may be discussed along lines of mutations or duplications of genes for co-activating or co-repressing nuclear proteins active at different levels within the circuits.

Thr176 regulates the activity of the mouse nuclear receptor CAR and is conserved in the NR1I subfamily members PXR and VDR

The mouse nuclear receptor CAR (constitutively active receptor) is a transcription factor that is activated by phenobarbital-type inducers such as TCPOBOP {1,4 bis[2-(3,5-dichloropyridyloxy)]benzene} in liver in vivo. However, CAR is constitutively active in cell-based transfection assays, the molecular mechanism for which has not been elucidated yet. In the model structure of CAR, Thr176 constitutes a part of the ligand-binding surface, but its side chain is not directed toward the surface, instead it forms a hydrogen bond with Thr350 in the AF2 (activation function 2) domain of CAR. Thr350 is known to regulate CAR activity [Ueda, Kakizaki, Negishi, and Sueyoshi (2002) Mol. Pharmacol. 61, 1284-1288]. Thr176 was mutated to various amino acids to examine whether this interaction played a role in conferring the constitutive activity. Hydrophobic and positively charged amino acids at position 176 abrogated the constitutive activity, whereas polar and negatively charged amino acids retained it. When one of the small hydrophobic amino acids, such as alanine or valine, was substituted for threonine, the mutants were fully activated by TCPOBOP. The co-activator SRC-1 (steroid receptor co-activator-1) regulated the activity changes associated with the mutations. Thr248 and Ser230 are the Thr176-corresponding residues in human pregnane X receptor and mouse vitamin D3 receptor respectively, interacting directly with the conserved threonine in the AF2 domains. Thr248 and Ser230 also regulated the ligand-dependent activity of these receptors by augmenting binding of the receptors to SRC-1. Thr176, Thr248 and Ser230 are conserved residues in the NR1I (nuclear receptor 1I) subfamily members and determine their activity.

Comparison of proposed frameworks for grouping polychlorinated biphenyl congener data applied to a case–control pilot study of prostate cancer

Although the commercial synthesis of polychlorinated biphenyls (PCBs) has been banned in the United States for several decades, they are persistent in the environment with exposure mainly being through diet. The biologic and toxic effects of PCBs and their metabolites are due in part to their ability to interact with several cellular and nuclear receptors, thereby altering signaling pathways and gene transcription. These effects include endocrine modulation and disruption. Therefore, the natural history of cancer in tissues expressing these receptors may be modulated by PCB congeners, which are known to have estrogenic, antiestrogenic, and other hormonal effects. Several frameworks for grouping PCB congeners based on these interactions have been proposed. We conducted a hospital-based, case–control pilot study of 58 prostate cancer cases and 99 controls to evaluate the association between the proposed PCB groupings and the risk of prostate cancer. Serum samples were analyzed for a total of 30 PCBs. In multivariate analyses, the odds of prostate cancer among men with the highest concentrations of moderately chlorinated PCBs or PCBs with phenobarbital-like activities (constitutively active receptor (CAR) agonists) was over two times that among men with the lowest concentrations. Increasing trends in risk across the concentration levels were also observed. These results suggest that a higher burden of PCBs that are CAR agonists may be positively associated with an increased risk of prostate cancer and they encourage further research in this area.

Transcriptional Determination of Sexually Dimorphic Expression of Nuclear Receptor Constitutively Active Receptor (CAR) in Wistar Rats

The mammalian constitutively active receptor (CAR) is a ligand-activated transcription factor that participates in controlling the expression of cytochrome P450 2B (CYP2B) genes in response to phenobarbital (PB) in rodents. CYP2B forms are highly inducible in liver and intestine in male Wistar rats. In contrast, PB-dependent increases in CYP2B activity are rarely observed in female Wistar rats. The profiles of CAR mRNA expression measured by RT-PCR in various organs in male and female Wistar rats were consistent with those of PB-dependent expression of CYP2B genes. The sex-related dimorphic PB-responses of CYP2B genes in Wistar rats might be determined transcriptionally as are the organospecific PB-responses in both males and females.

Effects of triaryl phosphates on mouse and human nuclear receptors

The constitutively active receptor (CAR) is a crucial regulator of genes encoding for enzymes active in drug/steroid oxidation, conjugation, and transport. In our attempt to isolate the endogenous inhibitory ligand(s) for the mouse CAR, we found surprisingly that the inhibitory activity was associated with di- and tri-isopropylated phenyl phosphates that were present in livers of untreated mice. Trans-activation experiments in mammalian cells with synthetic compounds verified that mouse CAR was inhibited by various isopropylated phenyl phosphates (40–80%). Such triaryl phosphates are widely used as fire retardants, lubricants, and plasticizers, and some of them are known to disturb reproduction by currently unknown mechanisms. Equipped with the finding that these compounds could interact with mouse CAR, we proceeded to determine their functional effects on other nuclear receptors. Human CAR and pregnane X receptor (PXR) were variably activated (2–5-fold) by triaryl phosphates while mouse PXR, peroxisome proliferator-activated receptor-α, and vitamin D receptor were refractory. Among steroid hormone receptors, the human androgen receptor was inhibited by triphenyl phosphate and di- ortho-isopropylated phenyl phosphate (40–50%) and activated by di- and tri- para-substituted phenyl phosphates (2-fold). Our results add to the list of CAR and PXR activators and suggest steroid-dependent biological pathways that may contribute to the reproductive effects of triaryl phosphates.

Role of the Defective Splicing of mRNA in the Lack of Pulmonary Expression of Constitutively Active Receptor in Rat

The mammalian constitutively active receptor (CAR) is a ligand-activated transcription factor that participates in controlling the expression of cytochrome P450 2B (CYP2B) genes in response to xenobiotics in an organ-specific manner. In the presence of phenobarbital (PB) or PB-type agents, hepatic CYP2B forms are highly inducible. In contrast, PB-dependent increases in the expression of CYP2B activities are rarely observed in the lung. Mature CAR mRNA could be detected in the liver of 7-week-old Wistar rats by RT-PCR, while lung CAR mRNA had a 91 bp extra nucleotide sequence inserted in a coding region of hepatic CAR mRNA. By comparing the full- length sequence of hepatic CAR mRNA, including 5′- and 3′-untranslated region (3′-UTR), with the genomic sequence completed in the present study, the genomic structure was clarified to consist of 9 exons and 8 introns, in which the coding region expanded from exon 2 in close to its 5′-end to the first one-third of exon 9 after 159 bp of 5′-UTR in the most frequently obtained cDNA clones. In pulmonary CAR mRNA, intron 6 was not spliced out, implying that the lack of CAR in the lung might in part result from the incomplete splicing of precursor mRNA during its maturation.

Species-specific responses of constitutively active receptor (CAR)–CYP2B coupling: lack of CYP2B inducer-responsive nuclear translocation of CAR in marine teleost, scup ( Stenotomus chrysops)

The mammalian constitutively active receptor (CAR) is a novel ligand-activated transcription factor that participates in controlling the expression of cytochrome P450 2B (CYP2B) genes in response to pharmaceutical agents (phenobarbital) and halogenated aromatic hydrocarbons ( ortho-substituted PCBs). The occurrence and physiological function of this protein are as yet unknown in marine animals, where there has been a paradoxical lack of induction by PB-type chemicals. One approach to understanding CAR function is to study the evolutionary history of processes such as CAR–CYP2B coupling. In this study, CAR function was evaluated in a representative teleost fish (scup, Stenotomus chrysops). Treatment of scup with 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), which is one of the most potent CYP2B inducers in mouse, caused no increase in hepatic alkoxyresorufin O-dealkylase activity nor in immunodetectable CYP2B-like protein levels. Western blot analyses of scup livers using anti-human CAR antisera revealed the occurrence of a putative CAR homologue in nuclear and cytoplasmic fractions, but no nuclear accumulation of CAR following TCPOBOP treatment, which is a first step regulating the transcriptional activation of CYP2B genes in mouse. Immunohistochemical study also showed no translocation of CAR into nucleus in the hepatocytes of TCPOBOP-treated scup. These results suggest that there may be species-specific differences in CAR activation or CAR–CYP2B coupling signaling transduction in fish from those in mouse.

Estrogen activation of the nuclear orphan receptor CAR (constitutive active receptor) in induction of the mouse Cyp2b10 gene.

The nuclear orphan receptor CAR (constitutively active receptor or constitutive androstane receptor) can be activated in response to xenochemical exposure, such as activation by phenobarbital of a response element called NR1 found in the CYP2B gene. Here various steroids were screened for potential endogenous chemicals that may activate CAR, using the NR1 enhancer and Cyp2b10 induction in transfected HepG2 cell and/or in mouse primary hepatocytes as the experimental criteria. 17beta-Estradiol and estrone activated NR1, whereas estriol, estetrol, estradiol sulfate, and the synthetic estrogen diethylstilbestrol did not. On the other hand, progesterone and androgens repressed NR1 activity in HepG2 cells, and the repressed NR1 activity was fully restored by estradiol. Moreover, estrogen treatment elicited nuclear accumulation of CAR in the mouse livers, as well as primary hepatocytes, and induced the endogenous Cyp2b10 gene. Ovariectomy did not affect either the basal or induced level of CAR in the nucleus of the female livers, while castration slightly increased the basal and greatly increased the induced levels in the liver nucleus of male mice. Thus, endogenous estrogen appears not to regulate CAR in female mice, whereas endogenous androgen may be the repressive factor in male mice. Estrogen at pharmacological levels is an effective activator of CAR in both female and male mice, suggesting a biological and/or toxicological role of this receptor in estrogen metabolism. In addition to mouse CAR, estrogens activated rat CAR, whereas human CAR did not respond well to the estrogens under the experimental conditions.

Phenobarbital-responsive nuclear translocation of the receptor CAR in induction of the CYP2B gene.

The constitutively active receptor (CAR) transactivates a distal enhancer called the phenobarbital (PB)-responsive enhancer module (PBREM) found in PB-inducible CYP2B genes. CAR dramatically increases its binding to PBREM in livers of PB-treated mice. We have investigated the cellular mechanism of PB-induced increase of CAR binding. Western blot analyses of mouse livers revealed an extensive nuclear accumulation of CAR following PB treatment. Nuclear contents of CAR perfectly correlate with an increase of CAR binding to PBREM. PB-elicited nuclear accumulation of CAR appears to be a general step regulating the induction of CYP2B genes, since treatments with other PB-type inducers result in the same nuclear accumulation of CAR. Both immunoprecipitation and immunohistochemistry studies show cytoplasmic localization of CAR in the livers of nontreated mice, indicating that CAR translocates into nuclei following PB treatment. Nuclear translocation of CAR also occurs in mouse primary hepatocytes but not in hepatocytes treated with the protein phosphatase inhibitor okadaic acid. Thus, the CAR-mediated transactivation of PBREM in vivo becomes PB responsive through an okadaic acid-sensitive nuclear translocation process.