Purpose: This study is aimed at investigating antineoplastic efficacy of histone deacetylase inhibitor (HDACI) LBH589 on renal cell carcinoma (RCC) and elucidating the novel molecular mechanisms involved in growth arrest and apoptosis by targeting the important nonhistone molecules. Experimental Design: We analyzed the growth-inhibitory effect of LBH589 on RCC by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in vitro and antitumor efficacy by xenograft experiments in vivo. To verify the associated molecular mechanisms involved in LBH589-mediated cell death and cell cycle progression by Western blotting and fluorescence-activated cell sorting analysis. Results: HDACI LBH589 induced degradation of both Aurora A and B kinases through a proteasome-mediated pathway by targeting HDAC3 and HDAC6.The dual degradation of Aurora A and B kinases mediated by LBH589 resulted in inducing G 2 -M arrest and apoptosis of renal cancer cell lines and our results also showed that LBH589 potently inhibited renal cancer cell growth in vitro and suppressed tumor formation in vivo. The Aurora A and B kinases and HDAC3 are overexpressed in the human RCC tumor tissues examined, which make them perfect targets for HDACI LBH589 treatment. Conclusions: Our in vitro and in vivo data showed that LBH589 has potent anticancer effect of renal cancer cells. LBH589 and other HDACI treatment resulted in inducing G 2 -M arrest and apoptosis of renal cancer cells through degradation of Aurora A and B kinases by inhibition of HDAC3 and HDAC6. The clinical efficacy of LBH589 in the treatment of patients with metastatic RCC, especially those with high Aurora kinase and HDAC expression, is worthy of further investigation.

The synaptonemal complex (SC) is a meiosis-specific tripartite structure that forms between two homologous chromosomes; it consists of a central region and two parallel lateral elements. Lateral elements also are called axial elements prior to synapsis. In Saccharomyces cerevisiae, Red1, Hop1, and Mek1 are structural components of axial/lateral elements. The red1/mek1/hop1 mutants all exhibit reduced levels of interhomolog recombination and produce no viable spores. Red1 is a phosphoprotein. Several earlier reports proposed that phosphorylated Red1 plays important roles in meiosis, including in signaling meiotic DNA damage or in preventing exit from the pachytene chromosomes. We report here that the phosphorylation of Red1 is carried out in CDC28-dependent and CDC28-independent manners. In contrast to previous results, we found Red1 phosphorylation to be independent of meiotic DNA recombination, the Mec1/Tel1 DNA damage checkpoint kinases, and the Mek1 kinase. To functionally validate the phosphorylation of Red1, we mapped the phosphorylation sites on this protein. A red1 14A mutant showing no detectable Red1 phosphorylation did not exhibit decreased sporulation efficiency, defects in viable spore production, or defects in meiotic DNA damage checkpoints. Thus, our results suggest that the phosphorylation of Red1 is not essential for its functions in meiosis.Meiosis is a critical component in the cycle of sexual reproduction, because it reduces the chromosome complement to haploidy in preparation for fertilization. This event is achieved by a single round of premeiotic DNA replication followed by two successive rounds of chromosome segregation to produce four haploid gametes. The first nuclear division (MI) is reductional, separating the newly recombined homologs from one another while leaving sister chromatids attached. The second nuclear division (MII), in which the sister chromatids segregate, is more typical of mitotic division and is called equational division. A prominent feature of meiosis is that pairing and recombination must occur between homologous chromosomes during the meiotic prophase. In contrast, these events rarely happen in mitosis. Meiotic DNA recombination plays a crucial role in meiosis, not only providing a potent source of genetic variation but also playing a mechanical role during MI. Specifically, crossover recombination results in a physical connection (i.e., chiasmata) between homologous chromosomes that allows them to orient properly on the spindle (for a review, see reference 59).Meiotic DNA recombination is initiated by the formation of DNA double-strand breaks (DSBs). Spo11, a meiosis-specific type II topoisomerase, generates DSBs together with several other factors in a cell cycle-programmed manner (26). The Mre11-Rad50-Xrs2 nuclease complex then resects these DSBs to generate 3Ј single-stranded tails that invade the intact DNA duplexes used for DNA repair (35). Most of these events use homologous chromosomes, not sister chromatids, as the templates for DNA repair to yield crossove...

The deregulation of epigenetics was involved in early and subsequent carcinogenic events. Reversing cancer epigenetics to restore a normal epigenetic condition could be a rational approach for cancer treatment and specialized prevention. In the present study, we found that the expression levels of two epigenetic markers, histone H3K27 trimethylation (H3K27me3), was low but histone H3S10 phosphorylation (pH3Ser10) was high in human bladder cancer tissues, which showed opposite expression patterns in their normal counterparts. Thus, we investigated whether a natural product, emodin, has the ability to reverse these two epigenetic modifications and inhibit bladder cancer cell growth. Emodin significantly inhibited the cell growth of four bladder cancer cell lines in a dose- and time-dependent manner. Emodin treatment did not induce specific cell cycle arrest, but it altered epigenetic modifications. Emodin treatment resulted in the suppression of pH3Ser10 and increased H3K27me3, contributing to gene silencing in bladder cancer cells. Microarray analysis demonstrated that oncogenic genes including fatty acid binding protein 4 (FABP4) and fibroblast growth factor binding protein 1 (HBP17), RGS4, tissue inhibitor of metalloproteinase 3 (TIMP3), WNT5b, URB, and collagen, type VIII, alpha 1 (COL8A1) responsible for proliferation, survival, inflammation, and carcinogenesis were significantly repressed by emodin. The ChIP assays also showed that emodin increased H3K27me3 but decreased pH3Ser10 modifications on the promoters of repressed genes, which indicate that emodin reverses the cancer epigenetics towards normal epigenetic situations. In conclusion, our work demonstrates the significant anti-neoplastic activity of emodin on bladder cancer cells and elucidates the novel mechanisms of emodin-mediated epigenetic modulation of target genes. Our study warrants further investigation of emodin as an effective therapeutic or preventive agent for bladder cancer.

Mitochondria are the powerhouses of cells. Mitochondrial CRaf is a potential cancer therapeutic target, as it regulates mitochondrial function and is localized to the mitochondria by its Nterminal domain. However, Raf inhibitor monotherapy can induce S338 phosphorylation of C-Raf (pC-Raf S338 ) and impede therapy. This study identified the interaction of C-Raf with S308 phosphorylated DAPK (pDAPK S308 ), which together became colocalized in the mitochondria to facilitate mitochondrial remodeling. Combined use of the Raf inhibitors sorafenib and GW5074 had synergistic anticancer effects in vitro and in vivo, but targeted mitochondrial function, rather than the canonical Raf signaling pathway. C-Raf depletion in knockout MEF C-RafÀ/À or siRNA knockdown ACHN renal cancer cells abrogated the cytotoxicity of combination therapy. Crystal structure simulation showed that GW5074 bound to C-Raf and induced a C-Raf conformational change that enhanced sorafenib-binding affinity.In the presence of pDAPK S308 , this drug-target interaction compromised the mitochondrial targeting effect of the N-terminal domain of C-Raf, which induced two-hit damages to cancer cells. First, combination therapy facilitated pC-Raf S338 and pDAPK S308 translocation from mitochondria to cytoplasm, leading to mitochondrial dysfunction and reactive oxygen species (ROS) generation. Second, ROS facilitated PP2A-mediated dephosphorylation of pDAPK S308 to DAPK. PP2A then dissociated from the C-Raf-DAPK complex and induced profound cancer cell death. Increased pDAPK S308 modification was also observed in renal cancer tissues, which correlated with poor disease-free survival and poor overall survival in renal cancer patients. Besides mediating the anticancer effect, pDAPK S308 may serve as a predictive biomarker for Raf inhibitors combination therapy, suggesting an ideal preclinical model that is worthy of clinical translation. Cancer Res; 75(17); 3568-82. Ó2015 AACR.