The discovery of novel 3-aryl-indazole derivatives as peripherally restricted pan-Trk inhibitors for the treatment of pain.

The design, synthesis, and biological evaluation of novel 3-aryl-indazole derivatives as peripherally selective pan-Trk inhibitors are described. Three strategies were used to obtain a potent compound exhibiting low central nervous system (CNS) penetration and high plasma exposure: 1) a structure-based drug design (SBDD) approach was used to improve potency; 2) a substrate for an efflux transporter for lowering brain penetration was explored; and 3) the most basic pKa (pKa-MB) value was used as an indicator to identify compounds with good membrane permeability. This enabled the identification of the peripherally targeted 17c with the potency, kinase-selectivity, and plasma exposure required to demonstrate in vivo efficacy in a Complete Freund's adjuvant (CFA)-induced thermal hypersensitivity model.

Estrogen Exhibits a Biphasic Effect on Prostate Tumor Growth through the Estrogen Receptor ß-KLF5 Pathway.

Estrogens are effective in the treatment of prostate cancer; however, the effects of estrogens on prostate cancer are enigmatic. In this study, we demonstrated that estrogen (17ß-estradiol [E2]) has biphasic effects on prostate tumor growth. A lower dose of E2 increased tumor growth in mouse xenograft models using DU145 and PC-3 human prostate cancer cells, whereas a higher dose significantly decreased tumor growth. We found that anchorage-independent apoptosis in these cells was inhibited by E2 treatment. Similarly, in vivo angiogenesis was suppressed by E2. Interestingly, these effects of E2 were abolished by knockdown of either estrogen receptor ß (ERß) or Krüppel-like zinc finger transcription factor 5 (KLF5). Ιn addition, E2 suppressed KLF5-mediated transcription through ERß, which inhibits proapoptotic FOXO1 and proangiogenic PDGFA expression. Furthermore, we revealed that a nonagonistic ER ligand GS-1405 inhibited FOXO1 and PDGFA expression through the ERß-KLF5 pathway and regulated prostate tumor growth without ERß transactivation. Therefore, these results suggest that E2 biphasically modulates prostate tumor formation by regulating KLF5-dependent transcription through ERß and provide a new strategy for designing ER modulators, which will be able to regulate prostate cancer progression with minimal adverse effects due to ER transactivation.

The nucleolar protein Myb-binding protein 1A (MYBBP1A) enhances p53 tetramerization and acetylation in response to nucleolar disruption.

Tetramerization of p53 is crucial to exert its biological activity, and nucleolar disruption is sufficient to activate p53. We previously demonstrated that nucleolar stress induces translocation of the nucleolar protein MYBBP1A from the nucleolus to the nucleoplasm and enhances p53 activity. However, whether and how MYBBP1A regulates p53 tetramerization in response to nucleolar stress remain unclear. In this study, we demonstrated that MYBBP1A enhances p53 tetramerization, followed by acetylation under nucleolar stress. We found that MYBBP1A has two regions that directly bind to lysine residues of the p53 C-terminal regulatory domain. MYBBP1A formed a self-assembled complex that provided a molecular platform for p53 tetramerization and enhanced p300-mediated acetylation of the p53 tetramer. Moreover, our results show that MYBBP1A functions to enhance p53 tetramerization that is necessary for p53 activation, followed by cell death with actinomycin D treatment. Thus, we suggest that MYBBP1A plays a pivotal role in the cellular stress response.

Nucleolar protein, Myb-binding protein 1A, specifically binds to nonacetylated p53 and efficiently promotes transcriptional activation.

Nucleolar dynamics are important for cellular stress response. We previously demonstrated that nucleolar stress induces nucleolar protein Myb-binding protein 1A (MYBBP1A) translocation from the nucleolus to the nucleoplasm and enhances p53 activity. However, the underlying molecular mechanism is understood to a lesser extent. Here we demonstrate that MYBBP1A interacts with lysine residues in the C-terminal regulatory domain region of p53. MYBBP1A specifically interacts with nonacetylated p53 and induces p53 acetylation. We propose that MYBBP1A dissociates from acetylated p53 because MYBBP1A did not interact with acetylated p53 and because MYBBP1A was not recruited to the p53 target promoter. Therefore, once p53 is acetylated, MYBBP1A dissociates from p53 and interacts with nonacetylated p53, which enables another cycle of p53 activation. Based on our observations, this MYBBP1A-p53 binding property can account for efficient p53-activation by MYBBP1A under nucleolar stress. Our results support the idea that MYBBP1A plays catalytic roles in p53 acetylation and activation.

MYBBP1A suppresses breast cancer tumorigenesis by enhancing the p53 dependent anoikis.

BACKGROUND: Tumor suppressor p53 is mutated in a wide variety of human cancers and plays a critical role in anoikis, which is essential for preventing tumorigenesis. Recently, we found that a nucleolar protein, Myb-binding protein 1a (MYBBP1A), was involved in p53 activation. However, the function of MYBBP1A in cancer prevention has not been elucidated. METHODS: Relationships between MYBBP1A expression levels and breast cancer progression were examined using patient microarray databases and tissue microarrays. Colony formation, xenograft, and anoikis assays were conducted using cells in which MYBBP1A was either knocked down or overexpressed. p53 activation and interactions between p53 and MYBBP1A were assessed by immunoprecipitation and western blot. RESULTS: MYBBP1A expression was negatively correlated with breast cancer tumorigenesis. In vivo and in vitro experiments using the breast cancer cell lines MCF-7 and ZR-75-1, which expresses wild type p53, showed that tumorigenesis, colony formation, and anoikis resistance were significantly enhanced by MYBBP1A knockdown. We also found that MYBBP1A binds to p53 and enhances p53 target gene transcription under anoikis conditions. CONCLUSIONS: These results suggest that MYBBP1A is required for p53 activation during anoikis; therefore, it is involved in suppressing colony formation and the tumorigenesis of breast cancer cells. Collectively, our results suggest that MYBBP1A plays a role in tumor prevention in the context of p53 activation.

Estrogen regulates tumor growth through a nonclassical pathway that includes the transcription factors ERß and KLF5.

Clinical evidence suggests that antiestrogens inhibit the development of androgen-insensitive prostate cancer. Here, we show that the estrogen receptor ß (ERß) mediates inhibition by the antiestrogen ICI 182,780 (ICI) and its enhancement by estrogen. ERß associated with gene promoters through the tumor-suppressing transcription factor KLF5 (Krüppel-like zinc finger transcription factor 5). ICI treatment increased the recruitment of the transcription coactivator CBP [CREB (adenosine 3',5'-monophosphate response element-binding protein)-binding protein] to the promoter of FOXO1 through ERß and KLF5, which enhanced the transcription of FOXO1. The increase in FOXO1 abundance led to anoikis in prostate cancer cells, thereby suppressing tumor growth. In contrast, estrogen induced the formation of complexes containing ERß, KLF5, and the ubiquitin ligase WWP1 (WW domain containing E3 ubiquitin protein ligase 1), resulting in the ubiquitination and degradation of KLF5. The combined presence of KLF5 and ERß positively correlated with longer cancer-specific survival in prostate cancer patients. Our results demonstrate that estrogens and antiestrogens affect prostate tumor growth through ERß-mediated regulation of KLF5.

PPARgamma ligands suppress the feedback loop between E2F2 and cyclin-E1.

PPARgamma is a nuclear hormone receptor that plays a key role in the induction of peroxisome proliferation. A number of studies showed that PPARgamma ligands suppress cell cycle progression; however, the mechanism remains to be determined. Here, we showed that PPARgamma ligand troglitazone inhibited G1/S transition in colon cancer cells, LS174T. Troglitazone did not affect on either expression of CDK inhibitor (p18) or Wnt signaling pathway, indicating that these pathways were not involved in the troglitazone-dependent cell cycle arrest. GeneChip and RT-PCR analyses revealed that troglitazone decreased mRNA levels of cell cycle regulatory factors E2F2 and cyclin-E1 whose expression is activated by E2F2. Down-regulation of E2F2 by troglitazone results in decrease of cyclin-E1 transcription, which could inhibit phosphorylation of Rb protein, and consequently evoke the suppression of E2F2 transcriptional activity. Thus, we propose that troglitazone suppresses the feedback loop containing E2F2, cyclin-E1, and Rb protein.