The TWEAK receptor Fn14 (TNFRSF12), a member of the TNF Receptor superfamily, can mediate many processes, including apoptosis. Fn14 agonists have therefore been the subject of interest as potential cancer therapeutics. In cell culture experiments, interferon gamma (IFNγ) is typically required for induction of apoptotic activity by either TWEAK or Fn14 agonistic antibodies in most cell lines. We have investigated the mechanism of IFNγ signaling and the role of JAK–STAT signaling in TWEAK/Fn14-mediated tumor cell killing. We found that IFNγ-mediated enhancement of tumor cell killing is JAK–STAT dependent, as JAK inhibitors block IFNγ−dependent TWEAK induced apoptosis. Exposure of tumor cells to IFNγ results in an increase in Fn14 expression on the cell surface, which may be a mechanism by which IFNγ induces sensitivity to TWEAK. In a reciprocal fashion, we observed that IFNγ receptor levels increase in response to TWEAK treatment in WiDr cells. Significantly, we found that TWEAK alone can induce STAT1 phosphorylation in WiDr tumor cells. Moreover, TWEAK induction of tumor cell apoptosis in WiDr cells in the absence of IFNγ is mediated by the JAK–STAT pathway. Correspondingly, we show that treatment of tumor bearing mice with mBIIB036, an Fn14 agonistic antibody, results in STAT1 phosphorylation in the tumors. Notably, the level of STAT1 phosphorylation appears to correlate with the degree of tumor growth inhibition by BIIB036 in vivo. Additionally, in WiDr cells, TWEAK induces a soluble factor, which we have identified as IFNβ, capable of independently inducing STAT1 phosphorylation when transferred to naïve cells. Finally, either IFNα or IFNβ can partially substitute for IFNγ in sensitizing tumor cells to Fn14 agonists. In summary, we show that TWEAK/Fn14 can signal through the JAK–STAT pathway to induce IFNβ, and that the ability of TWEAK to induce tumor cell apoptosis is mediated by JAK-STAT signaling. We also demonstrate that IFNγ enhancement of TWEAK/FN14-mediated tumor cell death is JAK-dependent and may occur by IFNγ-dependent upregulation of Fn14 on tumor cells. These findings may have implications for the appropriately targeted clinical development of Fn14 agonists as anti-cancer therapy.

Introduction: The development of new drugs over the last few decades has targeted specific proteins thought to be a key to the disease state. MAPK kinases 1 and 2 (commonly known as MEK1-2) represent such proteins as they lie downstream of important drug targets for oncology, such as EGFR, RAS and RAF. Several MEK1-2 inhibitors are currently in Phase I and II clinical trials in oncology. Areas covered: This review of current literature and recent conferences provides a background on the RAS-RAF-MEK-ERK signaling pathway and a discussion of early MEK inhibitors. The potential of MEK1-2 inhibitors for the treatment of inflammation is briefly presented. Preclinical and early clinical results are discussed for MEK inhibitors currently in development. Completed clinical trials of MEK inhibitors in oncology include some disappointments as well as some promising signs of the value of these compounds and we discuss the potential for MEK inhibitors as monotherapy and their use in drug combinations. Expert opinion: The utility of MEK inhibitors as anticancer agents will depend on careful patient selection based on the presence of mutations in genes such as KRAS and BRAF, the identification of additional predictive biomarkers, and an improved understanding of the benefit of drug combinations utilizing both established and emerging therapeutics.

Introduction: The development of new drugs over the last few decades has targeted specific proteins thought to be a key to the disease state. MAPK kinases 1 and 2 (commonly known as MEK1–2) represent such proteins as they lie downstream of important drug targets for oncology, such as EGFR, RAS and RAF. Several MEK1–2 inhibitors are currently in Phase I and II clinical trials in oncology. Areas covered: This review of current literature and recent conferences provides a background on the RAS-RAF-MEK-ERK signaling pathway and a discussion of early MEK inhibitors. The potential of MEK1–2 inhibitors for the treatment of inflammation is briefly presented. Preclinical and early clinical results are discussed for MEK inhibitors currently in development. Completed clinical trials of MEK inhibitors in oncology include some disappointments as well as some promising signs of the value of these compounds and we discuss the potential for MEK inhibitors as monotherapy and their use in drug combinations. Expert opinion: The utility of MEK inhibitors as anticancer agents will depend on careful patient selection based on the presence of mutations in genes such as KRAS and BRAF, the identification of additional predictive biomarkers, and an improved understanding of the benefit of drug combinations utilizing both established and emerging therapeutics.

Background Combining MEK inhibitors with other signalling pathway inhibitors or conventional cytotoxic drugs represents a promising new strategy against cancer. RDEA119/BAY 869766 is a highly potent and selective MEK1/2 inhibitor undergoing phase I human clinical trials. The effects of RDEA119/BAY 869766 as a single agent and in combination with rapamycin were studied in 3 early passage primary pancreatic cancer xenografts, OCIP19, 21, and 23, grown orthotopically. Methods Anti-cancer effects were determined in separate groups following chronic drug exposure. Effects on cell cycle and downstream signalling were examined by flow cytometry and western blot, respectively. Plasma RDEA119 concentrations were measured to monitor the drug accumulation in vivo . Results RDEA119/BAY 869766 alone or in combination with rapamycin showed significant growth inhibition in all the 3 models, with a significant decrease in the percentage of cells in S-phase, accompanied by a large decrease in bromodeoxyuridine labelling and cell cycle arrest predominantly in G1. The S6 ribosomal protein was inhibited to a greater extent with combination treatment in all the three models. Blood plasma pharmacokinetic analyses indicated that RDEA119 levels achieved in vivo are similar to those that produce target inhibition and cell cycle arrest in vitro . Conclusions Agents targeting the ERK and mTOR pathway have anticancer activity in primary xenografts, and these results support testing this combination in pancreatic cancer patients.

The present study was conducted to demonstrate of the immunohistochemical localization of vascular endothelial growth factor (VEGF) and its receptors (flt1/fms, flk1/KDR and flt4) as well as vascular endothelial growth inhibitor (VEGI) and to determine the correlation of VEGF and its receptors and VEGI with serum sex steroids (estrogen and progesterone) in the bovine uterus during the sexual cycle. The stage of the estrous cycle in 30 Holstein cattle was assessed based on the gross and histological appearance of the ovaries and uterus and on blood steroid hormone levels. Tissue samples obtained from the uterus were fixed in 10% formaldehyde for routine histological processing. During both follicular and luteal phases, positive cytoplasmic and membrane staining was achieved for VEGF and its receptors (flt1/fms, flk1/KDR and flt4) as well as VEGI in the luminal and glandular epithelial cells, the connective tissue and smooth muscle cells, and the vascular endothelial cells and smooth muscle cells in the uterus. The intensity, proportional and total scores determined for VEGF and its receptors (flt1/fms and flt4) as well as VEGI were greater in the luminal and glandular epithelial cells compared to the connective tissue and smooth muscle cells (P < 0.05). Furthermore, the number and intensity of the flk1/KDR positive cells were greater among the connective tissue cells compared to the luminal and glandular epithelial cells (P < 0.05). As a result, it was determined that the expression of VEGF and its receptors as well as VEGI in the bovine uterus during the follicular and luteal phases varied with different cell types. This suggests that depending on the stage of the sexual cycle, these factors may mediate the establishment of an appropriate environment for the nutritional supply and implantation of the embryo primarily due to the stimulation of angiogenesis but also through the increase in the secretory activity of the epithelial cells in the uterus. Furthermore, this indicates that ovarian steroid hormones play a significant role in regulating the expression of VEGF and its receptors as well as VEGI.

Although it is evident that androgens increase muscle mass and strength, little is known about the critical molecular targets of androgens in skeletal muscle. In rodents, the skeletal alpha-actin gene is a tissue-specific gene expressed only in the levator ani and other skeletal muscles but not in the prostate or preputial gland, the well-known androgen target tissue. We identified tissue-specific androgen-regulated genes in the skeletal muscle in rats after oral administration of androgens and focused on androgen-dependent up-regulation of the skeletal alpha-actin gene. To investigate the mechanism of action, an in vitro system with various cell lines and a series of deletion mutants of the alpha-actin promoter were used. The human skeletal alpha-actin promoter was activated by androgens in the muscle cell line C2C12 but not in the liver, prostate, or breast cancer cell lines in which exogenous human androgen receptor is expressed. The sequence of the promoter is sufficient for cell-specific androgen response, providing a model for the tissue specificity demonstrated in vivo. Using a series of deletion mutants, the androgen response can be maintained using just the proximal promoter region. The importance of androgen regulation of this small portion of the human skeletal alpha-actin promoter was demonstrated by the correlation between muscle and the alpha-actin promoter activity for an array of selective androgen receptor modulators (SARMs), including an orally active SARM LGD2226. Taken together, the results suggest that the regulation of skeletal alpha-actin by androgens/SARMs may represent an important model system for understanding androgen anabolic action in the muscle.

A number of conditions, including osteoporosis, frailty, and sexual dysfunction in both men and women have been improved using androgens. However, androgens are not widely used for these indications because of the side effects associated with these drugs. We describe an androgen receptor (AR) ligand that maintains expected anabolic activities with substantially diminished activity in the prostate. LGD2226 is a nonsteroidal, nonaromatizable, highly selective ligand for the AR, exhibiting virtually no affinity for the other intracellular receptors. We determined that AR bound to LGD2226 exhibits a unique pattern of protein-protein interactions compared with testosterone, fluoxymesterone (an orally available steroidal androgen), and other steroids, suggesting that LGD2226 alters the conformation of the ligand-binding domain. We demonstrated that LGD2226 is fully active in cell-based models of bone and muscle. LGD2226 exhibited anabolic activity on muscle and bone with reduced impact on prostate growth in rodent models. Biomechanical testing of bones from animals treated with LGD2226 showed strong enhancement of bone strength above sham levels. LGD2226 was also efficacious in a sex-behavior model in male rats measuring mounts, intromissions, ejaculations, and copulation efficiency. These results with an orally available, nonaromatizable androgen demonstrate the important role of the AR and androgens in mediating a number of beneficial effects in bone, muscle, and sexual function independent from the conversion of androgens into estrogenic ligands. Taken together, these results suggest that orally active, nonsteroidal selective androgen receptor modulators may be useful therapeutics for enhancing muscle, bone, and sexual function.

Nuclear receptors (NRs) are ligand-regulated transcription factors important in human physiology and disease. In certain NRs, including the androgen receptor (AR), ligand binding to the carboxy-terminal domain (LBD) regulates transcriptional activation functions in the LBD and amino-terminal domain (NTD). The basis for NTD-LBD communication is unknown but may involve NTD-LBD interactions either within a single receptor or between different members of an AR dimer. Here, measurement of FRET between fluorophores attached to the NTD and LBD of the AR established that agonist binding initiated an intramolecular NTD-LBD interaction in the nucleus and cytoplasm. This intramolecular folding was followed by AR self-association, which occurred preferentially in the nucleus. Rapid, ligand-induced intramolecular folding and delayed association also were observed for estrogen receptor-alpha but not for peroxisome proliferator activated receptor-gamma2. An antagonist ligand, hydroxyflutamide, blocked the NTD-LBD association within AR. NTD-LBD association also closely correlated with the transcriptional activation by heterologous ligands of AR mutants isolated from hormone-refractory prostate tumors. Intramolecular folding, but not AR-AR affinity, was disrupted by mutation of an alpha-helical ((23)FQNLF(27)) motif in the AR NTD previously described to interact with the AR LBD in vitro. This work establishes an intramolecular NTD-LBD conformational change as an initial component of ligand-regulated NR function.

Early studies in murine T cell lines indicated that transcriptional transactivation functions encoded in the glucocorticoid receptor (GR) N-terminal domain are required for glucocorticoid-mediated apoptosis. However, more recent studies in human T cell lines have suggested that the N-terminal domain is not necessary for steroid-regulated apoptosis and that GR-mediated transrepression may be the more critical mechanism. To better understand the contribution of the GR N-terminal transactivation domain in mediating murine thymocyte apoptosis, we stably transfected GR, GR variants, and the androgen receptor (AR) into receptor-negative S49 murine thymoma cells. GR expression levels were shown to be rate-limiting for initiating the apoptotic pathway, and a positive correlation between steroid sensitivity and GR-mediated induction of an integrated mouse mammary tumor virus (MMTV) LTR reporter gene was observed. Analysis of GR chimeric receptors containing the potent VP16 and E1A viral transactivation domains in place of the GR N terminus revealed that even low level expression of these receptors resulted in both enhanced steroid sensitivity and MMTV induction, thus supporting a role for transactivation in apoptosis. In contrast, we found that AR can initiate apoptosis in S49 cells after treatment with 5 alpha-dihydrotestosterone, despite its relative inability to induce high level expression of MMTV. To investigate this further, we examined the steroid-regulated expression of an endogenous thymocyte-specific gene called GIG18. We found that GIG18 was rapidly induced to comparable levels by both AR and GR, demonstrating that AR can indeed function as a transcriptional activator in S49 cells and, moreover, that GIG18 induction may be a marker of early apoptotic events in steroid-treated cells. Taken together, these results support our conclusion that transcriptional transactivation is a necessary signaling component of S49 cell apoptosis, although an additional role for GR-mediated transrepression cannot be excluded.

Transcriptional control of steroid-regulated gene networks by nuclear receptor proteins results in the coordinate expression of a limited number of target genes. Although much is known about the structure and function of steroid receptors, relatively few cell-specific steroid-regulated genes have been isolated and characterized. In this paper we describe results using mRNA differential display reverse transcriptase PCR (DDPCR) to identify and isolate short cDNA sequence tags from thymocyte and prostate cells under various hormone conditions. Using this technique we have isolated several differentially expressed sequence tags (DESTs) from the mouse thymocyte cell line WEHI 7.2. Two of these DESTs, GIG10 and GIG18, are rapidly induced by dexamethasone within 2 h of treatment. GIG10 is a novel sequence and GIG18 is the mouse homologue of a human expressed sequence tag isolated from activated B lymphocytes. We also used DDPCR to isolate DESTs from androgen-modulated rat ventral prostate tissue, one of which we characterized and found to correspond to the 3′ end of prostatic spermine binding protein mRNA, a known androgen-regulated gene. Modifications of the original DDPCR protocol, which we found can potentially decrease the frequency of isolating false-positive DESTs, are described and the merits of DDPCR, relative to other differential cDNA cloning strategies, are discussed.