Anti-androgen flutamide suppresses hepatocellular carcinoma cell proliferation via the aryl hydrocarbon receptor mediated induction of transforming growth factor-ß1.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and a member of the basic helix-loop-helix PER/ARNT/SIM family of chemosensors and developmental regulators. The AhR is widely known as a mediator of dioxin toxicity; however, it also suppresses cancer cell proliferation and recent findings have implicated its role as a tumor suppressor. We conducted a chemical library screen to identify nontoxic AhR ligands with anti-cancer effects and discovered flutamide (Eulexin) as a putative AhR ligand. Flutamide is an androgen receptor (AR) antagonist approved by the United States Food and Drug Administration for the treatment of prostate cancer. We found that flutamide inhibited the growth of several cancer cell lines independent of AR status, and that suppression of AhR expression reversed the anti-proliferative effects of flutamide. We investigated the AhR-dependent mechanism of action of flutamide in human hepatocellular carcinoma cells and identified that transforming growth factor-ß1 (TGF-ß1) is induced by flutamide in an AhR-dependent manner. In contrast, the potent AhR agonist 2,3,7,8-Tetrachlorodibenzo-p-dioxin had no effect on TGF-ß1 expression, indicating the ligand specificity of AhR activation. We also determined that TGF-ß1 induction is required for the AhR-dependent growth inhibitory effects of flutamide. Therefore, flutamide may be effective in AhR-positive cancers that are sensitive to TGF-ß1 signaling, such as hepatocellular carcinoma.

The aryl hydrocarbon receptor mediates raloxifene-induced apoptosis in estrogen receptor-negative hepatoma and breast cancer cells.

Identification of new molecular targets for the treatment of breast cancer is an important clinical goal, especially for triple-negative breast cancer, which is refractory to existing targeted treatments. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known primarily as the mediator of dioxin toxicity. However, the AhR can also inhibit cellular proliferation in a ligand-dependent manner and act as a tumor suppressor in mice, and thus may be a potential anticancer target. To investigate the AhR as an anticancer target, we conducted a small molecule screen to discover novel AhR ligands with anticancer properties. We identified raloxifene, a selective estrogen receptor (ER) modulator currently used in the clinic for prevention of ER-positive breast cancer and osteoporosis in post-menopausal women, as an AhR activator. Raloxifene directly bound the AhR and induced apoptosis in ER-negative mouse and human hepatoma cells in an AhR-dependent manner, indicating that the AhR is a molecular target of raloxifene and mediates raloxifene-induced apoptosis in the absence of ER. Raloxifene selectively induced apoptosis of triple-negative MDA-MB-231 breast cancer cells compared with non-transformed mammary epithelial cells via the AhR. Combined with recent data showing that raloxifene inhibits triple-negative breast cancer xenografts in vivo (Int J Oncol. 43(3):785-92, 2013), our results support the possibility of repurposing of raloxifene as an AhR-targeted therapeutic for triple-negative breast cancer patients. To this end, we also evaluated the role of AhR expression on survival of patients diagnosed with breast cancer. We found that higher expression of the AhR is significantly associated with increased overall survival and distant metastasis-free survival in both hormone-dependent (ER-positive) and hormone-independent (ER and progesterone receptor (PR)-negative) breast cancers. Together, our data strongly support the possibility of using the AhR as a molecular target for the treatment of hormone-independent breast cancers.

Regulation of Nur77 nuclear export by c-Jun N-terminal kinase and Akt.

Proapoptotic nuclear receptor family member Nur77 translocates from the nucleus to the mitochondria, where it interacts with Bcl-2 to trigger apoptosis. Nur77 translocation is induced by certain apoptotic stimuli, including the synthetic retinoid-related 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN)/CD437 class. In this study, we investigated the molecular mechanism by which AHPN/CD437 analog (E)-4-[3-(1-adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC) induces Nur77 nuclear export. Our results demonstrate that 3-Cl-AHPC effectively activated Jun N-terminal kinase (JNK), which phosphorylates Nur77. Inhibition of JNK activation by a JNK inhibitor suppressed 3-Cl-AHPC-induced Nur77 nuclear export and apoptosis. In addition, several JNK upstream activators, including the phorbol ester TPA, anisomycin and MAPK kinase kinase-1 (MEKK1), phosphorylated Nur77 and induced its nuclear export. However, Nur77 phosphorylation by JNK, although essential, was not sufficient for inducing Nur77 nuclear export. Induction of Nur77 nuclear export by MEKK1 required a prolonged MEKK1 activation and was attenuated by Akt activation. Expression of constitutively active Akt prevented MEKK1-induced Nur77 nuclear export. Conversely, transfection of dominant-negative Akt or treatment with a phosphatidylinositol 3-kinase (PI3-K) inhibitor accelerated MEKK1-induced Nur77 nuclear export. Furthermore, mutation of an Akt phosphorylation residue Ser351 in Nur77 abolished the effect of Akt or the PI3-K inhibitor. Together, our results demonstrate that both activation of JNK and inhibition of Akt play a role in translocation of Nur77 from the nucleus to the cytoplasm.

Novel target genes of the Ah (dioxin) receptor: transcriptional induction of N-myristoyltransferase 2.

Dioxins are potent mammalian carcinogens and toxins affecting liver, skin, and immune and reproductive systems. The intracellular Ah receptor, a ligand-dependent transcription factor of the basic region/helix-loop-helix/Per-Ahr/Arnt-Sim homology domain (bHLH-PAS) protein family, mediates responses to dioxins. Target genes of the Ah receptor that mediate dioxin toxicity and carcinogenicity are, however, mostly unknown. We used 5L rat hepatoma cells to identify dioxin-inducible genes by suppression subtractive hybridization. Eleven cDNA fragments were identified that represented novel sequences or genes for which induction by dioxins had not been known. N-myristoyltransferase 2 (NMT2) is one of the later dioxin-inducible genes. Induction of NMT2 was confirmed in livers of mice in vivo. NMT2 induction was a direct consequence of Ah receptor activation in 5L cells. [(3)H]myristic acid incorporation into 5L cell proteins was inducible by dioxins, indicating that protein myristoylation is a regulated rather than a housekeeping function and that NMT activity is limiting in noninduced 5L cells. Here we show for the first time that expression of NMT2 and induced protein myristoyltransferase activity are direct responses to carcinogen exposure. Because inappropriate protein NH(2)-terminal myristoylation appears to play a role in carcinogenesis, induction of NMT2 may play a central role in dioxin carcinogenicity.

Apoptosis induction in cancer cells by a novel analogue of 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid lacking retinoid receptor transcriptional activation activity.

The retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN) is reported to have anticancer activity in vivo. Induction of cell cycle arrest and apoptosis in cancer cell lines refractory to standard retinoids suggests a retinoid-independent mechanism of action for AHPN. Conformational studies suggested that binding of AHPN does not induce an unusual conformation in retinoic acid receptor (RAR) gamma. The 3-chloro AHPN analogue MM11453 inhibited the growth of both retinoid-resistant (HL-60R leukemia, MDA-MB-231 breast, and H292 lung) and retinoid-sensitive (MCF-7 breast, LNCaP prostate, and H460 lung) cancer cell lines by inducing apoptosis at similar concentrations. Before apoptosis, MM11453 induced transcription factor TR3 expression and loss of mitochondrial membrane potential characteristic of apoptosis. MM11453 lacked the ability to significantly activate RARs and retinoid X receptor alpha to initiate (TREpal)(2)-tk-CAT reporter transcription. These results, differential proteolysis-sensitivity assays, and glutathione S-transferase-pulldown experiments demonstrate that, unlike AHPN or the natural or standard synthetic retinoids, MM11453 does not behave as a RAR or retinoid X receptor alpha transcriptional agonist. These studies strongly suggest that AHPN exerts its cell cycle arrest and apoptotic activity by a signaling pathway independent of retinoid receptor activation.

Cytochrome c release and apoptosis induced by mitochondrial targeting of nuclear orphan receptor TR3.

TR3, an immediate-early response gene and an orphan member of the steroid-thyroid hormone-retinoid receptor superfamily of transcription factors, regulates apoptosis through an unknown mechanism. In response to apoptotic stimuli, TR3 translocates from the nucleus to mitochondria to induce cytochrome c release and apoptosis. Mitochondrial targeting of TR3, but not its DNA binding and transactivation, is essential for its proapoptotic effect. Our results reveal a mechanism by which a nuclear transcription factor translocates to mitochondria to initiate apoptosis.

Unique anti-activator protein-1 activity of retinoic acid receptor beta.

The anticancer effects of retinoids are mainly mediated by two classes of nuclear receptors, the retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which are encoded by three distinct genes (alpha, beta, and gamma). Recent studies have demonstrated that RARbeta plays a critical role in mediating anticancer effects of retinoids. However, how RARbeta exerts its potent anticancer effects remains largely unknown. In this study, we investigated anti-Activator Protein-1 (AP-1) activity of RARbeta. In a transient transfection assay, all three RAR subtypes, RARalpha, RARbeta, and RARgamma, could effectively inhibit phorbol ester 12-O-tetradecanoylphorbol-13-acetate-induced AP-1 activity and the activity of oncogenes c-Jun and c-Fos on AP-1 containing reporter genes in the presence of retinoic acid (RA). However, RARbeta showed a strong RA-independent inhibition of AP-1 activity, whereas inhibition of AP-1 activity by RARalpha and RARgamma was RA dependent. By using several hybrid receptors that contain either the COOH-terminal portion or the NH2-terminal portion of RARbeta, we demonstrated that the NH2-terminal portion of RARbeta, the A/B domain, was mainly responsible for the RA-independent inhibition of AP-1 activity. This activity was not attributable to constitutive AF-1 activity of RARbeta, because it did not activate several RA response element-containing reporter genes. In addition, inhibition of histone deacetylase activity by trichostatin A did not overcome the inhibitory effect of RARbeta. In cancer cells, stable transfection of RARbeta exhibited strong inhibition of AP-1 activity, even in the absence of RA. Moreover, expression of endogenous AP-1-responsive gene collagenase I was strongly repressed in cancer cells stably transfected with RARbeta. In studying the antitransforming activity of RARbeta, we observed that the growth of breast cancer MDA-MB231 cells in soft agar was significantly repressed in a RA-independent manner when cells were stably transfected with RARbeta but not RARalpha. Together, our results demonstrate that RARbeta may exert its potent anticancer effect in part through its unique anti-AP-1 activity.

Orphan receptor COUP-TF is required for induction of retinoic acid receptor beta, growth inhibition, and apoptosis by retinoic acid in cancer cells.

Retinoic acid receptor beta (RARbeta) plays a critical role in mediating the anticancer effects of retinoids. Expression of RARbeta is highly induced by retinoic acid (RA) through a RA response element (betaRARE) that is activated by heterodimers of RARs and retinoid X receptors (RXRs). However, RARbeta induction is often lost in cancer cells despite expression of RARs and RXRs. In this study, we provide evidence that orphan receptor COUP-TF is required for induction of RARbeta expression, growth inhibition, and apoptosis by RA in cancer cells. Expression of COUP-TF correlates with RARbeta induction in a variety of cancer cell lines. In addition, stable expression of COUP-TF in COUP-TF-negative cancer cells restores induction of RARbeta expression, growth inhibition, and apoptosis by RA, whereas inhibition of COUP-TF by expression of COUP-TF antisense RNA represses the RA effects. In a transient transfection assay, COUP-TF strongly induced transcriptional activity of the RARbeta promoter in a RA- and RARalpha-dependent manner. By mutation analysis, we demonstrate that the effect of COUP-TF requires its binding to a DR-8 element present in the RARbeta promoter. The binding of COUP-TF to the DR-8 element synergistically increases the RA-dependent RARalpha transactivation function by enhancing the interaction of RARalpha with its coactivator CREB binding protein. These results demonstrate that COUP-TF, by serving as an accessory protein for RARalpha to induce RARbeta expression, plays a critical role in regulating the anticancer activities of retinoids.

p27(Kip1) induction and inhibition of proliferation by the intracellular Ah receptor in developing thymus and hepatoma cells.

The Ah receptor (AhR), a bHLH/PAS transcription factor, mediates dioxin toxicity in the immune system, skin, testis and liver. Toxic phenomena are associated with altered cell proliferation or differentiation, but signaling pathways of AhR in cell cycle regulation are poorly understood. Here we show that AhR induces the p27(Kip1) cyclin/cdk inhibitor by altering Kip1 transcription in a direct mode without the need for ongoing protein synthesis or cell proliferation. This is the first example of Kip1 being a direct transcriptional target of a toxic agent that affects cell proliferation. Kip1 causes dioxin-induced suppression of 5L hepatoma cell proliferation because Kip1 antisense-expressing cells are resistant to dioxins. Kip1 is also induced by dioxins in cultures of fetal thymus glands concomitant with inhibition of proliferation and severe reduction of thymocyte recovery. Kip1 expression is likely to mediate these effects as thymic glands of Kip1-deficient mice (Kip1(Delta51)) are largely, though not completely, resistant.

Mapping of epitopes on porcine zona pellucida-3 alpha by monoclonal antibodies inhibiting oocyte-sperm interaction.

Zona pellucida glycoprotein 3 alpha (ZP3 alpha) has been designated as the primary sperm receptor ligand in porcine gamete interaction. In this study, epitopes were mapped on porcine ZP3 alpha (pZP3 alpha) by using monoclonal antibodies (mAbs) possessing in vitro contraceptive efficacy. Using Western blots, we tested recombinant pZP3 alpha fragments expressed as fusion proteins in Escherichia coli and corresponding to pZP3 alpha precursor protein amino acid residues 18-142 (F1), 140-243 (F2), 239-363 (F3), and 359-462 (F4), for reactivities with mAbs. MAb-403 reacted with F3, and mAbs-412 and -421 with F2. MAb-420 showed weak reactivity with F1. Synthesis of overlapping 12-mer peptides further resolved the epitope for mAb-420 to amino acid residues 133-144, mAb-421 to 157-168, mAb-412 to 205-216, and mAb-403 to 301-312. MAbs-412 and -420 inhibited the binding of boar sperm to zona-encased porcine oocytes. These results, the first to define peptide epitopes of pZP3 alpha, should assist in the design of a synthetic peptide-based immunocontraceptive vaccine.

Complementation of Ah receptor deficiency in hepatoma cells: negative feedback regulation and cell cycle control by the Ah receptor.

The Ah receptor (AhR) is a ligand-dependent transcription factor subunit that heterodimerizes with the AhR nuclear translocator (Arnt) and mediates the predominant biological effects of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD activates target genes in xenobiotica metabolism in many cell lines and, more specifically, delays G1-S progression of 5L hepatoma cells. Here we describe transient and stable AhR-expression analysis in AhR-deficient subclones of the TCDD-sensitive 5L cells. We tested the integrity of the AhR-signaling system beyond the lack of the receptor in the variant subclone and analyzed the role of AhR in cell cycle regulation. Transiently expressed AhR has a high basal activity on promoters containing AhR-binding sites, so-called XREs, when transfected into receptor-deficient variant cells compared to wild-type cells. Single- and double-hybrid analysis dissociates AhR ligand responsiveness, transactivation, and heterodimerization with Arnt from receptor binding to an XRE. Hybrid receptors also show the high basal activity in the absence of exogenous TCDD in AhR-deficient variant cells, indicating that the endogenous AhR-activating signal acts directly on the receptor rather than XRE-dependent promoters or DNA binding of the receptor. Stable expression of AhR in variant cell clones by retroviral infection fully reconstitutes TCDD responsiveness, including target-gene induction and delay of cell cycle progression. These AhR-reconstituted cells, like AhR-containing wild-type cells, show low basal activity of the transiently expressed AhR hybrid. Thus, the increased basal activity in AhR-deficient cells suggests a negative feedback control of AhR activity. In vitro ligand-binding assays are compatible with the idea that the increased basal activity is due to the accumulation of an AhR-binding endogenous ligand. In conclusion, AhR is causally responsible for TCDD-dependent cell cycle regulation and feedback control of AhR activity.

Nucleotide sequence of cDNA encoding bonnet monkey (Macaca radiata) zona pellucida glycoprotein-ZP3.

The cDNA encoding bonnet monkey zona pellucida ZP3 from bonnet ovary has been amplified by polymerase chain reaction. The ZP3 gene has an open reading frame of 1272 nucleotides encoding a polypeptide of 424 amino acid residues which shares 93.9% overall identity with human ZP3. Bonnet ZP3 has four potential attachment sites for N-linked sugar chains which are also conserved in human ZP3. Bonnet ZP3 has 14 cysteine residues compared with 15 in human ZP3. The highest disparity between these molecules was restricted to a domain represented by amino acid residues 370-398. These results have important implications for the use of bonnet monkey as an animal model for evaluation and development of contraceptive vaccine based on ZP3 for human use.