Kosuke Mizutani

CCR1/CCL5 interaction promotes invasion of taxane-resistant PC3 prostate cancer cells by increasing secretion of MMPs 2/9 and by activating ERK and Rac signaling

Castration-refractory prostate cancer (CRPC) is treated with taxane-based chemotherapy, but eventually becomes drug resistant. It is thus essential to identify novel therapeutic targets for taxane resistance in CRPC patients. We investigated the role of the chemokine (C-C motif) receptor 1 (CCR1) and its ligand, chemokine (C-C motif) ligand 5 (CCL5), in taxane-resistant CRPC using paclitaxel-resistant prostate cancer cells (PC3PR) established from PC3 cells. We found that the expression levels of CCR1 mRNA and protein were up-regulated in PC3PR cells compared to PC3 cells. In order to investigate the role of increased CCR1 in PC3PR cells, we stimulated cells with CCL5, one of the chemokine ligands of CCR1. In CCL5-stimulated PC3PR cells, siRNA-mediated knockdown of CCR1 expression reduced phosphorylation of ERK1/2 and Rac1/cdc42. Furthermore, CCR1 knockdown and MEK1/2 inhibition decreased CCL5-stimulated secretion of MMPs 2 and 9, which play important roles in cancer cell invasion and metastasis. In the Matrigel invasion assay, knockdown of CCR1 and inhibition of the ERK and Rac signaling pathways significantly decreased the number of invading cells. Finally, the serum CCL5 protein level as measured by ELISA was not different among the three groups of patients: those with negative prostate biopsy, those at initial diagnosis of prostate cancer, and those with taxane-resistant prostate cancer. These results demonstrated for the first time that the interaction of CCR1 with CCL5 caused by increased expression of CCR1 promotes invasion of PC3PR cells by increasing secretion of MMPs 2 and 9 and by activating ERK and Rac signaling. Our findings suggest that CCR1 could be a novel therapeutic target for taxane-resistant CRPC.

Sept8 controls the binding of vesicle-associated membrane protein 2 to synaptophysin.

Septins, a conserved family of GTP/GDP‐binding proteins, are present in organisms as diverse as yeast and mammals. We analyzed the distribution of five septins, Sept6, Sept7, Sept8, Sept9 and Sept11, in various rat tissues by western blot analyses and found all septins to be expressed in brain. We also examined the developmental changes of expression of these septins in the rat brain and found that the level of Sept8 increased during post‐natal development. Morphological analyses revealed that Sept8 is enriched at pre‐synapses. Using yeast two‐hybrid screening, we identified vesicle‐associated membrane protein 2 (VAMP2), a soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE), as an interacting protein for Sept8. Synaptophysin is reported to associate with and recruit VAMP2 to synaptic vesicles and dissociate prior to forming the SNARE complex consisting of VAMP2, syntaxin and synaptosome‐associated protein of 25 kDa. We showed that Sept8 suppresses the interaction between VAMP2 and synaptophysin through binding to VAMP2. In addition, we found that Sept8 forms a complex with syntaxin1A, and the Sept8‐VAMP2 interaction is disrupted by synaptosome‐associated protein of 25 kDa. These results suggest that Sept8 may participate in the process of the SNARE complex formation and subsequent neurotransmitter release.

Bacterial loads of Ureaplasma urealyticum contribute to development of urethritis in men.

Ureaplasma urealyticum could be a pathogen of non-gonococcal urethritis (NGU) in men. However, ureaplasma is often detected in men without NGU, and the proportion of cases possibly attributable to this pathogen is still undefined. We attempted to determine the bacterial loads of U. urealyticum significantly associated with NGU. The 16S rRNA genes of U. urealyticum were quantified by a real-time polymerase chain reaction-based assay in first-void urine (FVU) from 26 asymptomatic and 25 symptomatic men positive for U. urealyticum. The leucocyte counts in first-void urine (FVU) were determined as an objective measure of inflammatory response to ureaplasma in the hosts by automated quantitative urine particle analysis. Positive correlations were observed between copies of the 16S rRNA genes of U. urealyticum per ml and the leucocyte counts per µl in FVU (r = 0.49, p = 0.0003). Loads of ≥104 copies of the 16S rRNA gene of U. urealyticum/ml, corresponding to ≥5 × 103 cells of U. urealyticum/ml in FVU, were significantly associated with the presence of urethritis symptoms (p < 0.0001) and with higher leukocyte counts in FVU (p < 0.0001). The bacterial load of U. urealyticum, possibly of ≥5 × 103 cells of U. urealyticum/ml in FVU, could be significantly associated with the development of symptomatic NGU.

Serum exosomal P-glycoprotein is a potential marker to diagnose docetaxel resistance and select a taxoid for patients with prostate cancer

OBJECTIVES Docetaxel is used as the first-line chemotherapy for castration-resistant prostate cancer (CRPC), but docetaxel resistance occurs in part owing to induction of P-glycoprotein (P-gp) encoded by multidrug resistance protein 1 (MDR1) gene. A recently developed taxane-cabazitaxel-has poor affinity for P-gp and is thereby effective in docetaxel-resistant CRPC. It has been recently demonstrated that exosomes in the body fluids could serve as a diagnostic marker because they contain proteins and RNAs specific to the cells from which they are derived. In this study, we aimed to investigate if P-gp in blood exosomes could be a marker to diagnose docetaxel resistance and select a taxoid for patients with CRPC. METHODS AND MATERIALS Exosomes were isolated by differential centrifugation from docetaxel-resistant prostate cancer (PC-3) cells (PC-3R) and their parental PC-3 cells and from the serum of patients. Silencing of P-gp was performed by small interfering RNA transfection. Protein expression was examined by Western blot analysis. Viability of cells treated with docetaxel or cabazitaxel was determined by water soluble tetrazolium salt (WST) assay. RESULTS The level of P-gp was higher in exosomes as well as cell lysates from PC-3R cells than in those from PC-3 cells. Cabazitaxel effectively killed PC-3R cells, and MDR1 knockdown improved the sensitivity of PC-3R cells to docetaxel but not to cabazitaxel. The P-gp level in blood exosomes was relatively higher in clinically docetaxel-resistant patients than in therapy-naïve patients. CONCLUSIONS Our results suggest that detection of P-gp in blood exosomes, which is involved in resistance to docetaxel but not to cabazitaxel, could be useful to diagnose docetaxel resistance and select an appropriate taxoid for patients with CRPC-docetaxel or cabazitaxel.

Phosphorylation by extracellular signal‐regulated kinase of a multidomain adaptor protein, vinexin, at synapses

Vinexin is an adaptor protein that is supposed to play pivotal roles in cell adhesion, cytoskeletal organization and signaling. At least three splice variants, vinexinα, β and γ, have so far been reported. In spite of the possible importance of vinexin, the properties and functions of vinexin in neuronal cells are almost unknown. Here we show that vinexin isoforms are expressed in rat brain in a developmental stage‐dependent manner, and that vinexinα is relatively abundant in the telencephalon regions of the adult rat brain. An immunohistochemical study showed the localization of vinexinα in neurons and glia in the rat brain. In primary cultured rat hippocampal neurons, vinexin was found to be present at synapses and filopodia in growth cones by immunofluorescent analyses. Biochemical fractionation revealed the distribution of vinexin in synaptosomes. Nerve terminal localization of vinexin was confirmed by electron microscopy. Vinexinβ is reported to be phosphorylated by extracellular signal‐regulated kinase (ERK) at Ser189, which is equivalent to Ser593 of vinexinα. We thus constructed a site‐ and phosphorylation state‐specific antibody to monitor the ERK‐mediated phosphorylation of vinexin. In immunofluorescent analyses, the phosphorylation was observed at synapses formed among cultured rat hippocampal neurons and it was reduced by treatment of the cells with PD98059. In an immunoelectron microscopic examination, the phosphorylation signal was mainly detected on the postsynaptic side of synapses in the rat hippocampal neurons. As active ERK was co‐localized with vinexin in synapses, the ERK signal is likely to be involved in the regulation of vinexin‐dependent cellular processes in synapses. On the other hand, the phosphorylation was hardly detected in neurons cultured for 3 days, suggesting the presence of a yet unidentified regulatory mechanism of vinexin at the growth cone.

Essential roles of ERK-mediated phosphorylation of vinexin in cell spreading, migration and anchorage-independent growth

Vinexin is an adaptor protein supposed to play pivotal roles in various cellular events such as cell adhesion, cytoskeletal organization, signaling and gene expression. Despite the possible importance, physiological functions and regulatory mechanisms of vinexin are largely unknown. In addition, although vinexin was reported to be phosphorylated by extracellular signal-regulated kinase (ERK), physiological significance of the phosphorylation remains to be elucidated. Here we carried out characterization of endogenous vinexin and found that it was enriched at the leading edge of migrating cells and focal adhesions of spread cells. In the analyses using ERK-phosphorylated vinexin-specific antibody, the phosphorylation signal was also detected at the leading edges of migrating cells and at cell periphery of spreading cells, whereas only faint signal was observed at focal adhesions of well-spread cells. We then established LNCaP cell lines stably expressing GFP-fused vinexinβ or two mutants at Ser189 that mimic the ERK-phosphorylated or -unphosphorylated vinexinβ. Based on the analyses using the lines, the phosphorylation was likely to inhibit the cell spreading and migration. On the other hand, anchorage-independent cell growth was inhibited by unphosphorylated vinexinβ. Taken together, ERK-mediated phosphorylation of vinexinβ is strongly suggested to occur in a spatio-temporally regulated manner and play important roles in cell spreading, migration and anchorage-independent growth.

miR-130a activates apoptotic signaling through activation of caspase-8 in taxane-resistant prostate cancer cells

The acquisition of drug resistance is one of the most malignant phenotypes of cancer and identification of its therapeutic target is a prerequisite for the development of novel therapy. MicroRNAs (miRNAs) have been implicated in various types of cancer and proposed as potential therapeutic targets for patients. In the present study, we aimed to identify miRNA that could serve as a therapeutic target for taxane‐resistant prostate cancer.

Integrin β4 and vinculin contained in exosomes are potential markers for progression of prostate cancer associated with taxane-resistance

Treatment with taxanes for castration-resistant prostate cancer often leads to the development of resistance. It has been recently demonstrated that exosomes present in the body fluids contain proteins and RNAs in the cells from which they are derived and could serve as a diagnostic marker for various diseases. In the present study, we aimed to identify proteins contained in exosomes that could be markers for progression and taxane-resistance of prostate cancer. Exosomes were isolated by differential centrifugation from the culture medium of taxane-resistant human prostate cancer PC-3 cells (PC-3R) and their parental PC-3 cells. Isolated exosomes were subjected to iTRAQ-based quantitative proteomic analysis. Exosomes were also isolated from the culture medium by using anti-CD9 antibody-conjugated magnetic beads. Protein expression was knocked down by siRNA transfection followed by analysis of the silencing effects. Proteomic analysis showed that integrin β4 (ITGB4) and vinculin (VCL) were upregulated in exosomes derived from PC-3R cells compared to PC-3 cells. The elevation of ITGB4 and VCL was confirmed in exosomes captured by anti-CD9 antibody from the culture medium of PC-3R cells. Silencing of ITGB4 and VCL expression did not affect proliferation and taxane-resistance of PC-3R cells, but ITGB4 knockdown attenuated both cell migration and invasion and VCL knockdown reduced invasion. Our results suggest that ITGB4 and VCL in exosomes could be useful markers for progression of prostate cancer associated with taxane-resistance, providing the basis for development of an exosome-based diagnostic system.

A Comparative Study of Protein Profiling by Proteomic Analysis in Camptothecin-Resistant PC3 and Camptothecin-Sensitive LNCaP Human Prostate Cancer Cells

Introduction: Drug resistance is a major obstacle for the therapy of prostate cancer, but its underlying mechanisms are not clarified. To detect some candidate marker proteins which may confer resistance to the anticancer drug camptothecin (CPT; DNA topoisomerase 1 inhibitor), the current study deals with the comparative proteomic profiling of CPT-resistant PC3 and CPT-sensitive LNCaP human prostate cancer cell lines which have been widely employed as a useful model to investigate prostate cancer cells. Materials and Methods: The global profiling of the protein expression was investigated in CPT-resistant PC3 and CPT-sensitive LNCaP prostate cancer cells using 2-dimensional polyacrylamide gel electrophoresis/matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Results: 144 proteins were identified and their expression levels were compared between the two cell lines. Four proteins – annexin A1, glutathione-S-transferase (GST) π, galectin (Gal) 3 and glucose-regulated protein 78/Bip – that are suggested to contribute to the development of drug resistance were found to be preferentially or highly expressed in PC3 cells, whereas LNCaP cells did not show detectable expression of annexin A1, GST-π and Gal-3. Conclusion: The expression level of these proteins and/or mRNAs could be a useful parameter to evaluate the chemotherapy resistance in clinical specimens of prostate cancer.

Possible interaction of a Rho effector, Rhotekin, with a PDZ-protein, PIST, at synapses of hippocampal neurons.

Rhotekin, an effector of Rho, is highly expressed in the brain but its function is almost unknown. In an attempt to define the properties of Rhotekin in neuronal cells, we focused on its interaction with polarity-related molecules. In the present study, we raised the possibility that Rhotekin interacts with a PDZ-protein, PIST (PDZ domain protein interacting specifically with TC10) in vitro, and found that these proteins form complex in the rat brain tissues. We then demonstrated that Rhotekin and PIST are expressed in developmental stage-specific manners in the rat brain. In immunofluorescence analyses, Rhotekin and PIST were suggested to co-localize at synapses in rat primary cultured hippocampal neurons. On the other hand, PIST was found to form immunocomplex with another PDZ-binding protein, beta-catenin, in HEK293 cells and in the rat brain, and co-localized with this protein at dendritic filopods. The interaction of PIST with beta-catenin was inhibited by the presence of Rhotekin. These results suggest a possible yet unidentified role of Rhotekin, in harmony with PIST and beta-catenin, in neuronal cells.