Silencing and reactivation of the selective estrogen receptor modulator-estrogen receptor alpha complex

4-Hydroxytamoxifen (4-OHT), a selective estrogen receptor modulator, is an agonist at a transforming growth factor-alpha (TGF-alpha) target gene in situ in MDA-MB-231 human breast cancer cells stably transfected with wild-type human ERalpha. In contrast, raloxifene (Ral) is a complete antiestrogen silencing activation function (AF) 1 and AF2 in this system. A natural mutation D351YERalpha enhances 4-OHT agonist activity and changes Ral-like compounds from antagonists to partial agonists. We reasoned that: either the conformation of the Ral-D351YERalpha is altered, thereby reactivating AF2 in the ligand binding domain, or the change at amino acid 351 allosterically reactivates AF1 in the Ral-D351YERalpha complex. Unlike the estradiol-ERalpha complex, agonist activity of 4-OHT and raloxifene through ERalpha and D351YERalpha were not attributed to coactivator (such as SRC-1, AIB1) binding to the ligand binding domain. We conclude that the classic AF2 is not responsible for the agonist activities of 4-OHT-ERalpha, 4-OHT-D351YERalpha, and Ral-D351YERalpha. To address the role of AF1, stable transfectants of ERalpha or D351YERalpha with an AF1 deletion (D351deltaAF1, D351YdeltaAF1) were generated in MDA-MB-231 cells. Additionally, D538A/E542A/D545A triple mutations within helix 12 (D351-3m, D351Y3m) or the COOH-terminal 537 deletion (D351delta537) were tested. The agonist activities of 4-OHT and raloxifene were lost in these stable transfectants, but antiestrogenic action was retained. The reactivation of an estrogen-like property of the Ral-ERalpha complex through AF1 with the D351Y mutation illustrates a novel allosteric mechanism for the selective estrogen receptor modulator ERalpha complex.     

Overexpression of androgen receptor enhances the binding of the receptor to the chromatin in prostate cancer

Androgen receptor (AR) is overexpressed in the majority of castration-resistant prostate cancers (CRPCs). Our goal was to study the effect of AR overexpression on the chromatin binding of the receptor and to identify AR target genes that may be important in the emergence of CRPC. We have established two sublines of LNCaP prostate cancer (PC) cell line, one overexpressing AR 2-3-fold and the other 4-5-fold compared with the control cells. We used chromatin immunoprecipitation (ChIP) and deep-sequencing (seq) to identify AR-binding sites (ARBSs). We found that the number of ARBSs and the AR-binding strength were positively associated with the level of AR when cells were stimulated with low concentrations of androgens. In cells overexpressing AR, the chromatin binding of the receptor took place in 100-fold lower concentration of the ligand than in control cells. We confirmed the association of AR level and chromatin binding in two PC xenografts, one containing AR gene amplification with high AR expression, and the other with low expression. By combining the ChIP-seq and expression profiling, we identified AR target genes that are upregulated in PC. Of them, the expression of ZWINT, SKP2 (S-phase kinase-associated protein 2 (p45)) and FEN1 (flap structure-specific endonuclease 1) was demonstrated to be increased in CRPC, while the expression of SNAI2 was decreased in both PC and CRPC. FEN1 protein expression was also associated with poor prognosis in prostatectomy-treated patients. Finally, the knock-down of FEN1 with small interfering RNA inhibited the growth of LNCaP cells. Our data demonstrate that the overexpression of AR sensitizes the receptor binding to chromatin, thus, explaining how AR signaling pathway is reactivated in CRPC cells.     

Somatic change in the estrogen receptor gene associated with altered expression of the progesterone receptor

Using a fragment of the estrogen receptor (ER) cDNA as a probe, Southern blots of HindIII-digested genomic DNA, from breast tumours and non-cancerous tissue, revealed a 1.6 kb hybridizing fragment (1.6KbHF) in 3 out of 30 tumours, and in 31 out of 80 mastectomy samples (16 sectors from each of 5 patients), which was significantly correlated with the absence of progesterone receptor. In 4 of the patients, sectors which showed the 1.6KbHF co-existed in the same breast with sectors which did not. Neither the use of higher concentrations of the restriction enzyme, not high salt extraction of DNA changed the abundance of the 1.6KbHF relative to other bands. Similar studies on EcoRI digests of the same DNA samples failed to show any variants. As a further control, hybridization of the same Southern blots of HindIII-digested DNA, with a cytochrome P450IIA3 cDNA, also failed to show variants in these samples. It is concluded that the presence of the 1.6KbHF represents a somatic change in the nucleus of these samples which may have functional importance.     

Spontaneous development of liver tumors in the absence of the bile acid receptor farnesoid X receptor

Farnesoid X receptor (FXR, NR1H4) is a member of the nuclear hormone receptor superfamily, which plays an essential role in regulating bile acid, lipid, and glucose homeostasis. Both male and female FXR(-/-) mice spontaneously developed liver tumors; however, no other tumors were developed after 15 months of age. In contrast, no liver tumors were observed in wild-type mice of the same age. Histologic analyses confirm that tumors were hepatocellular adenoma and carcinoma. Although there was no obvious tumor at ages 9 to 12 months, FXR(-/-) livers displayed prominent liver injury and inflammation. Strong labeling of apoptotic hepatocytes and liver damage-induced compensatory regeneration were observed. Deregulation of genes involved in bile acid homeostasis in FXR(-/-) mice was consistent with abnormal levels of bile acids presented in serum and liver. Genes involved in inflammation and cell cycle were up-regulated in aging FXR(-/-) mice but not in wild-type controls. Increasing the bile acid levels by feeding mice with a 0.2% cholic acid diet strongly promoted N-nitrosodiethylamine-initiated liver tumorigenesis, whereas lowering bile acid pool in FXR(-/-) mice by a 2% cholestyramine feeding significantly reduced the malignant lesions. Our results suggest an intriguing link between metabolic regulation and hepatocarcinogenesis.     

Targeting the epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) is a member of the erbB family of tyrosine kinase receptors (RTK). The EGFR is involved in cell proliferation, metastasis and angiogenesis, and is expressed in a large proportion of epithelial tumours. The two main classes of EGFR inhibitors in clinical trials are the RTK inhibitors and the monoclonal antibodies. The clinical development of EGFR inhibitors has introduced new challenges to the design of phase I, II, and III trials. Both classes of agents can be safely administered at doses sufficient to inhibit the EGFR system. Receptor tyrosine kinase inhibitors have been extensively evaluated in non-small-cell lung cancer. In this setting, gefitinib has demonstrated activity in patients who fail initial chemotherapy. Monoclonal antibodies have been developed in combination with cytotoxic chemotherapy in several tumour types, most notably colorectal and head and neck cancer. The preliminary results suggest an increase in response rate and time to progression with the combination of cetuximab and chemotherapy in both disease models. Future issues in the development of EGFR inhibitors include the identification of biologic predictors of response, combination with other targeted agents, and their utilisation in earlier stage malignancies.     

Cisplatin-induced activation of the EGF receptor

Cisplatin (CDDP) is an efficient DNA-damaging antitumor agent employed for the treatment of various human cancers. CDDP activates nuclear as well as cytoplasmatic signaling pathways involved in regulation of the cell cycle, damage repair and programmed cell death. Here we report that CDDP also activates a membrane-integrated protein, the epidermal growth factor receptor (EGFR). We show that EGFR is activated in response to CDDP in various types of cells that overexpress the receptor, including transformed human glioma cells and human breast tumor cells. CDDP-induced EGFR activation requires its kinase activity, as it can be blocked by an EGFR kinase inhibitor or by expression of a kinase dead receptor. We also show that CDDP-induced EGFR activation is independent of receptor ligand. CDDP induces the activation of c-Src, and EGFR activation is blocked by Src-family inhibitor PP1, suggesting that Src kinases mediate CDDP-induced EGFR activation. We propose that EGFR activation in response to CDDP is a survival response, since inhibition of EGFR activation enhances CDDP-induced death. These findings show that signals generated by DNA damage can modulate EGFR activity, and argue that interfering with CDDP-induced EGFR activation in tumor cells might be a useful approach to sensitize these cells to genotoxic agents.     

Ganglioside modulation of the PDGF receptor

Summary Gangliosides are a family of glycolipids that are present at the cell surface of all mammalian cells. Patterns of gangliosides are different in gliomas than normal brain, and exogenously added gangliosides affect the growth of cultured glioma cells. Gangliosides inhibit the activities of several kinases, including protein kinase C (PKC) and cAMP-kinase. U-1242 MG cells (derived from a human malignant glioma) have receptors for platelet-derived growth factor (PDGF) that become phosphorylated on tyrosine when exposed to PDGF. Exposure of these cells to PDGF also causes an increase in intracellular calcium concentration ([Ca²⁺]_i) and induces a translocation of PKC to the membrane. Preincubation of U-1242 MG cells with several species of gangliosides inhibits the increase in ([Ca²⁺]_i) and PKC translocation in response to PDGF, but GM3 is much less effective than other species tested. This is due to a lack of activation of the receptor tyrosine kinase as monitored by phosphorylation of the receptor on tyrosine residues, but is not due to an inhibition of binding of PDGF to its receptors. The lack of activation of the PDGF receptor tyrosine kinase is due to an inhibition of dimerization of the receptor monomers by gangliosides GM1, GM2, GD1a, GT1b, but not GM3. Therefore, gangliosides may be involved in coordinating the activities of multiple trophic factors simultaneously acting on a cell by regulating the dimerization of their respective receptor monomers.     

Diminished hepatocellular proliferation in mice humanized for the nuclear receptor peroxisome proliferator-activated receptor alpha

Lipid-lowering fibrate drugs function as agonists for the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha). Sustained activation of PPARalpha leads to the development of liver tumors in rats and mice. However, humans appear to be resistant to the induction of peroxisome proliferation and the development of liver cancer by fibrate drugs. The molecular basis of this species difference is not known. To examine the mechanism determining species differences in peroxisome proliferator response between mice and humans, a PPARalpha-humanized mouse line was generated in which the human PPARalpha was expressed in liver under control of the tetracycline responsive regulatory system. The PPARalpha-humanized and wild-type mice responded to treatment with the potent PPARalpha ligand Wy-14643 as revealed by induction of genes encoding peroxisomal and mitochondrial fatty acid metabolizing enzymes and resultant decrease of serum triglycerides. However, surprisingly, only the wild-type mice and not the PPARalpha-humanized mice exhibited hepatocellular proliferation as revealed by elevation of cell cycle control genes, increased incorporation of 5-bromo-2'-deoxyuridine into hepatocyte nuclei, and hepatomegaly. These studies establish that following ligand activation, the PPARalpha-mediated pathways controlling lipid metabolism are independent from those controlling the cell proliferation pathways. These findings also suggest that structural differences between human and mouse PPARalpha are responsible for the differential susceptibility to the development of hepatocarcinomas observed after treatment with fibrates. The PPARalpha-humanized mice should serve as models for use in drug development and human risk assessment and to determine the mechanism of hepatocarcinogenesis of peroxisome proliferators.     

Androgen receptor splice variants activating the full-length receptor in mediating resistance to androgen-directed therapy

Upregulation of constitutively-active androgen receptor splice variants (AR-Vs) has been implicated in AR-driven tumor progression in castration-resistant prostate cancer. To date, functional studies of AR-Vs have been focused mainly on their ability to regulate gene expression independent of the full-length AR (AR-FL). Here, we showed that AR-V7 and AR^v567es, two major AR-Vs, both facilitated AR-FL nuclear localization in the absence of androgen and mitigated the ability of the antiandrogen enzalutamide to inhibit AR-FL nuclear trafficking. AR-V bound to the promoter of its specific target without AR-FL, but co-occupied the promoter of canonical AR target with AR-FL in a mutually-dependent manner. AR-V expression attenuated both androgen and enzalutamide modulation of AR-FL activity/cell growth, and mitigated the in vivo antitumor efficacy of enzalutamide. Furthermore, AR^v567es levels were upregulated in xenograft tumors that had acquired enzalutamide resistance. Collectively, this study highlights a dual function of AR-Vs in mediating castration resistance. In addition to trans-activating target genes independent of AR-FL, AR-Vs can serve as a “rheostat” to control the degree of response of AR-FL to androgen-directed therapy via activating AR-FL in an androgen-independent manner. The findings shed new insights into the mechanisms of AR-V-mediated castration resistance and have significant therapeutic implications.     

Gastrin-releasing peptide receptor mediates activation of the epidermal growth factor receptor in lung cancer cells

Gastrin-releasing peptide receptor (GRPR) and the epidermal growth factor receptor (EGFR) are expressed in several cancers including non-small cell lung cancer (NSCLC). Here we demonstrate the activation of EGFR by the GRPR ligand, gastrin-releasing peptide (GRP), in NSCLC cells. GRP induced rapid activation of p44/42 MAPK in lung cancer cells through EGFR. GRP-mediated activation of MAPK in NSCLC cells was abrogated by pretreatment with the anti-EGFR-neutralizing antibody, C225. Pretreatment of NSCLC cells with neutralizing antibodies to the EGFR ligands, TGF-A or HB-EGF, also decreased GRP-mediated MAPK activation. On matrix metalloproteinase (MMP) inhibition, GRP failed to activate MAPK in NSCLC cells. EGF and GRP both stimulated NSCLC proliferation, and inhibition of either EGFR or GRPR resulted in cell death. Combining a GRPR antagonist with the EGFR tyrosine kinase inhibitor, gefitinib, resulted in additive cytotoxic effects. Additive effects were seen at gefitinib concentrations from 1 to 18 microM, encompassing the ID50 values of both gefitinib-sensitive and gefitinib-resistant NSCLC cell lines. Because a major effect of GRPR appears to be promoting the release of EGFR ligand, this study suggests that a greater inhibition of cell proliferation may occur by abrogating EGFR ligand release in consort with inhibition of EGFR.     

Interaction of the aryl hydrocarbon receptor ligand 6-methyl-1,3,8-trichlorodibenzofuran with estrogen receptor alpha

The polycyclic aromatic hydrocarbon 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) is related to the industrial byproduct dioxin and is a weak agonist and partial antagonist at the aryl hydrocarbon receptor (AhR). Tamoxifen is used for the treatment and prevention of breast cancer and interferes with the interaction of estrogen with estrogen receptor alpha (ER). The combination of MCDF and tamoxifen lowered the effective dose of both drugs required to inhibit 7,12-dimethylbenz(a)anthracene-induced mammary tumor growth in rats and protected against the estrogenic effects of tamoxifen on the uterus in rats (A. McDougal et al., Cancer Res 2001;61:3902-7), pointing to the potential use of MCDF in breast cancer treatment. Potential AhR-ER cross-talk is evidenced by the antiestrogenic activity of MCDF and the degradative effect of MCDF on ER protein levels. Our studies confirmed that MCDF degraded the ER. MCDF displayed antiestrogenic activity at higher concentrations in MCF-7 human breast cancer cells, but MCDF alone (10(-6) M) stimulated the growth of MCF-7 cells. MCDF also activated an estrogen response element (ERE)-luciferase reporter and increased mRNA levels of the estrogen-responsive gene transforming growth factor (TGF)-alpha. The estrogenic effects of MCDF are ER dependent because they were blocked by the pure antiestrogen ICI 182,780. MCDF induced ER-coactivator interaction in glutathione S-transferase pull-down assays and the formation of an ER.ERE complex in gel mobility shift assays, further indicating that the estrogenic actions of MCDF are mediated by the ER. In addition, knockdown of the AhR with small interfering RNA did not affect MCDF-induced ERE-luciferase activity. Overall, these data support the conclusion that MCDF is a partial agonist at the ER. This study provides the first evidence for the direct interaction of the ER with MCDF and challenges the view that MCDF is simply an AhR-specific ligand.     

Oncogenic and invasive potentials of human macrophage-stimulating protein receptor,the RON receptor tyrosine kinase

The product of the RON (recepteur d'origine nantais) gene belongs to the MET proto-oncogene family, a distinct subfamily of receptor tyrosine kinases. The ligand of RON was identified as macrophage-stimulating protein (MSP), a member of the plasminogen-related growth factor family. RON is mainly expressed in cells of epithelial origin and is required for embryonic development. In vitro RON activation results in epithelial cell dissociation, migration and matrix invasion, suggesting that RON might be involved in the pathogenesis of certain epithelial cancers in vivo. Indeed, recent studies have shown that RON expression is significantly altered in several primary human cancers, including those of the breast and colon. Truncation of the RON protein has also been found in primary tumors from the gastrointestinal tract. These alterations lead to constitutive activation of RON that causes cell transformation in vitro, induces neoplasm formation in athymic nude mice, and promotes tumor metastasis into the lung. Studies employing transgenic models further demonstrated that over-expression of RON in lung epithelial cells results in multiple tumor formation with features of large cell undifferentiated carcinoma. The oncogenic activities of RON are mediated by RON-transduced signals that promote unbalanced cell growth and transformation leading to tumor development. Thus, abnormal accumulation and activation of RON could play a critical role in vivo in the progression of certain malignant human epithelial cancers.     

Structural analysis of the ErbB-2 receptor using monoclonal antibodies: Implications for receptor signalling

The extracellular part of ErbB-2 is formed by 4 domains, specifically, L1, L2 that adopt a beta-helical structure and S1, S2 that consist of several cysteine-rich, EGF-fold modules. These ectodomains mediate ErbB-2 dimerisation with itself or with other members of the epidermal growth factor receptor (EGFR) family, events essential to both ErbB-2 signaling and the development of certain malignancies. The anti-ErbB-2 monoclonal antibodies N12, N28 and L87 bind to the polypeptides C531-A586, T216-C235 and C220-C235 respectively. In this study, glycine walking and random mutagenesis were used to further delineate the critical residues involved in antibody binding. A molecular model of ErbB-2 ectodomains was then constructed based on the recently published coordinates of the EGFR (EGFR) model. This model rationalized successfully many features of our epitope mapping, including their location in modules within the S1 and S2 domains and the importance of Arg545, Gln548 and Leu561 for N12 binding. Further investigation of the functional effects of the anti-ErbB-2 monoclonal antibodies demonstrated that N28 strongly stimulated ErbB-2 phosphorylation and MAPK activation whereas N12 had no effect. As bivalency is required for the action of these antibodies we propose that at least 2 different kinds of ErbB-2 homodimers can be formed as relative rotational isomers and that the S1 and S2 domains are instrumental in determining the relative orientations of the ErbB-2 homodimers, such that different signaling effects are induced.     

Transactivation of the epidermal growth factor receptor by formylpeptide receptor exacerbates the malignant behavior of human glioblastoma cells

The G protein-coupled formylpeptide receptor (FPR), which mediates leukocyte migration in response to bacterial and host-derived chemotactic peptides, promotes the chemotaxis, survival, and tumorigenesis of highly malignant human glioblastoma cells. Because glioblastoma cells may also express other receptors for growth signals, such as the epidermal growth factor (EGF) receptor (EGFR), we investigated the role of EGFR in the signaling cascade of FPR and how two receptors cross-talk to exacerbate tumor growth. We found that N-formyl-methionyl-leucyl-phenylalanine, an FPR agonist peptide, rapidly induced EGFR phosphorylation at tyrosine residue (Tyr) 992, but not residues 846, 1068, or 1173, in glioblastoma cells, whereas all these residues were phosphorylated after only EGF treatment. The FPR agonist-induced EGFR phosphorylation in tumor cells was dependent on the presence of FPR as well as Galphai proteins, and was controlled by Src tyrosine kinase. The transactivation of EGFR contributes to the biological function of FPR in glioblastoma cells because inhibition of EGFR phosphorylation significantly reduced FPR agonist-induced tumor cell chemotaxis and proliferation. Furthermore, depletion of both FPR and EGFR by short interference RNA abolished the tumorigenesis of the glioblastoma cells. Our study indicates that the glioblastoma-promoting activity of FPR is mediated in part by transactivation of EGFR and the cross-talk between two receptors exacerbates the malignant phenotype of tumor cells. Thus, targeting both receptors may yield antiglioblastoma agents superior to those targeting one of them.     

Modulation of the folate receptor alpha gene by the estrogen receptor: mechanism and implications in tumor targeting

The folate receptor (FR) type alpha is a promising target for diagnostic imaging agents and therapeutic intervention in major subtypes of gynecological malignancies; however, the receptor levels in the tumors are variable and are generally relatively low in estrogen receptor (ER)-positive tumors. Here we report that the FR-alpha gene promoter is repressed in the presence of 17beta-estradiol and derepressed by the antiestrogens tamoxifen and ICI 182780 in a promoter-specific and ER-alpha-dependent manner in carcinoma cell lines including HeLa (cervical carcinoma), BG-1 (ovarian carcinoma), and IGROV-1 (ovarian carcinoma). The ligand and ER dose response of the FR-alpha promoter and its time course paralleled those of a classical estrogen response element-mediated effect. Antiestrogens produced an ER-dependent increase of up to 36-fold in the expression of the endogenous FR-alpha gene. Deletion analysis and FR-alpha/SV40 promoter chimeras showed that the ER effect is mediated exclusively within the G/C-rich region in the TATA-less P4 promoter of FR-alpha; electrophoretic mobility shift analysis demonstrated interaction of ER at only one of three G/C-rich elements. ER-beta only modestly affected FR-alpha promoter activity but did not diminish the ER-alpha-mediated effects. The ER corepressor, SMRT, enhanced the repression by 17beta-estradiol/ER, but ER coactivators, including SRC family members, did not appreciably impact the ER ligand response. The results suggest that in ER+ tumors, FR-alpha expression is directly and actively suppressed and predict that a brief treatment with antiestrogens will boost FR-alpha expression by passive derepression, enhancing the efficacy of FR-targeted diagnostic and therapeutic applications. They also reveal novel aspects of gene repression by ER.     

The erbB gene and the EGF receptor

The epidermal growth factor (EGF) receptor is a plasma membrane glycoprotein. It contains four distinct segments: an N-terminal EGF binding domain which is exposed at the cell surface; a short transmembrane segment; a cytoplasmic domain with protein-tyrosine kinase activity; and a C-terminal regulatory segment. Binding of EGF to the external domain of the receptor activates the protein-tyrosine kinase activity of the receptor, and this elevated kinase activity is presumed to be involved in the activation of cell growth. The v-erbB transforming gene of avian erythroblastosis virus is derived, by retroviral transduction, from the gene (c-erbB) which encodes the avian EGF receptor. The transforming capacity of v-erbB appears to result from truncation of the receptor. In erythroid cells, truncation of the N-terminal ligand binding domain is sufficient for transformation, whereas in fibroblasts removal of an additional C-terminal segment is required for transformation. The EGF receptor is subject to complex regulatory controls, including ligand activation, downregulation by internalization, autophosphorylation and autoregulation and transmodulation involving phosphorylation by kinase C. This review is centered around the hypothesis that the transforming capacity of the truncated v-erbB gene product results from a loss in sensitivity to regulators and the consequent activation of protein kinase activity.