Discovery of Novel Bicyclic Pyrazoles as Potent PIP5K1C Inhibitors.

Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) is generated by phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) from phosphatidylinositol 4-phosphate (PI4P). Structurally diverse and selective inhibitors against PIP5Ks are required to further elucidate the therapeutic potential for PIP5K inhibition, although the effects of PIP5K inhibition on various diseases and their symptoms, such as cancer and chronic pain, have been reported. Our medicinal chemistry efforts led to novel and potent PIP5K1C inhibitors. Compounds 30 and 33 not only showed potent activity but also demonstrated low total clearance in mice and high levels of kinase selectivity. These compounds might serve as tools to further elucidate the complex biology and therapeutic potential of PIP5K inhibition.

Semaphorin 3 C enhances putative cancer stemness and accelerates peritoneal dissemination in pancreatic cancer.

PURPOSE: Semaphorins, axon guidance cues in neuronal network formation, have been implicated in cancer progression. We previously identified semaphorin 3 C (SEMA3C) as a secreted protein overexpressed in pancreatic ductal adenocarcinoma (PDAC). We, therefore, hypothesized that SEMA3C supports PDAC progression. In this study, we aimed to investigate the clinical features of SEMA3C, especially its association with chemo-resistance and peritoneal dissemination. METHODS: In resected PDAC tissues, we assessed the relationship between SEMA3C expression and clinicopathological features by immunohistochemistry. In vitro studies, we have shown invasion assay, pancreatosphere formation assay, colony formation assay, cytotoxicity assay, and activation of SEMA3C downstream targets (c-Met, Akt, mTOR). In vivo, we performed a preclinical trial to confirm the efficacy of SEMA3C shRNA knockdown and Gemcitabine and nab-Paclitaxel (GnP) in an orthotopic transplantation mouse model and in peritoneal dissemination mouse model. RESULTS: In resected PDAC tissues, SEMA3C expression correlated with invasion and peritoneal dissemination after surgery. SEMA3C promoted cell invasion, self-renewal, and colony formation in vitro. We further demonstrated that SEMA3C knockdown increased Gem-induced cytotoxicity by suppressing the activation of the Akt/mTOR pathway via the c-Met receptor. Combination therapy with SEMA3C knockdown and GnP reduced tumor growth and peritoneal dissemination. CONCLUSIONS: SEMA3C enhances peritoneal dissemination by regulating putative cancer stemness and Gem resistance and activates phosphorylation of the Akt/mTOR pathway via c-Met. Our findings provide a new avenue for therapeutic strategies in regulating peritoneal dissemination during PDAC progression.

Prosaposin, tumor-secreted protein, promotes pancreatic cancer progression by decreasing tumor-infiltrating lymphocytes.

Glycoproteins produced by tumor cells are involved in cancer progression, metastasis, and the immune response, and serve as possible therapeutic targets. Considering the dismal outcomes of pancreatic ductal adenocarcinoma (PDAC) due to its unique tumor microenvironment, which is characterized by low antitumor T-cell infiltration, we hypothesized that tumor-derived glycoproteins may serve as regulating the tumor microenvironment. We used glycoproteomics with tandem mass tag labeling to investigate the culture media of three human PDAC cell lines, and attempted to identify the key secreted proteins from PDAC cells. Among the identified glycoproteins, prosaposin (PSAP) was investigated for its functional contribution to PDAC progression. PSAP is highly expressed in various PDAC cell lines; however, knockdown of intrinsic PSAP expression did not affect the proliferation and migration capacities. Based on the immunohistochemistry of resected human PDAC tissues, high PSAP expression was associated with poor prognosis in patients with PDAC. Notably, tumors with high PSAP expression showed significantly lower CD8+ T-cell infiltration than those with low PSAP expression. Furthermore, PSAP stimulation decreased the proportion of CD8+ T cells in peripheral blood monocytes. Finally, in an orthotopic transplantation model, the number of CD8+ T cells in the PSAP shRNA groups was significantly increased, resulting in a decreased tumor volume compared with that in the control shRNA group. PSAP suppresses CD8+ T-cell infiltration, leading to the promotion of PDAC progression. However, further studies are warranted to determine whether this study contributes to the development of a novel immunomodulating therapy for PDAC.

C4b-binding protein α-chain enhances antitumor immunity by facilitating the accumulation of tumor-infiltrating lymphocytes in the tumor microenvironment in pancreatic cancer.

BACKGROUND: Recent studies indicate that complement plays pivotal roles in promoting or suppressing cancer progression. We have previously identified C4b-binding protein α-chain (C4BPA) as a serum biomarker for the early detection of pancreatic ductal adenocarcinoma (PDAC). However, its mechanism of action remains unclear. Here, we elucidated the functional roles of C4BPA in PDAC cells and the tumor microenvironment. METHODS: We assessed stromal C4BPA, the C4BPA binding partner CD40, and the number of CD8+ tumor-infiltrating lymphocytes in resected human PDAC tissues via immunohistochemical staining. The biological functions of C4BPA were investigated in peripheral blood mononuclear cells (PBMCs) and human PDAC cell lines. Mouse C4BPA (mC4BPA) peptide, which is composed of 30 amino acids from the C-terminus and binds to CD40, was designed for further in vitro and in vivo experiments. In a preclinical experiment, we assessed the efficacy of gemcitabine plus nab-paclitaxel (GnP), dual immune checkpoint blockades (ICBs), and mC4BPA peptide in a mouse orthotopic transplantation model. RESULTS: Immunohistochemical analysis revealed that high stromal C4BPA and CD40 was associated with favorable PDAC prognosis (P=0.0005). Stromal C4BPA strongly correlated with the number of CD8+ tumor-infiltrating lymphocytes (P=0.001). In in vitro experiments, flow cytometry revealed that recombinant human C4BPA (rhC4BPA) stimulation increased CD4+ and CD8+ T cell numbers in PBMCs. rhC4BPA also promoted the proliferation of CD40-expressing PDAC cells. By contrast, combined treatment with gemcitabine and rhC4BPA increased PDAC cell apoptosis rate. mC4BPA peptide increased the number of murine T lymphocytes in vitro and the number of CD8+ tumor-infiltrating lymphocytes surrounding PDAC tumors in vivo. In a preclinical study, GnP/ICBs/mC4BPA peptide treatment, but not GnP treatment, led to the accumulation of a greater number of CD8+ T cells in the periphery of PDAC tumors and to greater tumor regression than did control treatment. CONCLUSIONS: These findings demonstrate that the combination of GnP therapy with C4BPA inhibits PDAC progression by promoting antitumor T cell accumulation in the tumor microenvironment.

Inhibitory effects of ceramide kinase on Rac1 activation, lamellipodium formation, cell migration, and metastasis of A549 lung cancer cells.

Ceramide kinase (CerK) phosphorylates ceramide to ceramide-1-phosphate (C1P), a bioactive sphingolipid. Since the mechanisms responsible for regulating the proliferation and migration/metastasis of cancer cells by the CerK/C1P pathway remain unclear, we conducted the present study. The knockdown of CerK in A549 lung and MCF-7 breast cancer cells (shCerK cells) increased the formation of lamellipodia, which are membrane protrusions coupled with cell migration. Mouse embryonic fibroblasts prepared from CerK-null mice also showed an enhanced formation of lamellipodia. The overexpression of CerK inhibited lamellipodium formation in A549 cells. The knockdown of CerK increased the number of cells having lamellipodia with Rac1 and the levels of active Rac1-GTP form, whereas the overexpression of CerK decreased them. CerK was located in lamellipodia after the epidermal growth factor treatment, indicating that CerK functioned there to inhibit Rac1. The migration of A549 cells was negatively regulated by CerK. An intravenous injection of A549-shCerK cells into nude mice resulted in markedly stronger metastatic responses in the lungs than an injection of control cells. The in vitro growth of A549 cells and in vivo expansion after the injection into mouse flanks were not affected by the CerK knockdown. These results suggest that the activation of CerK/C1P pathway has inhibitory roles on lamellipodium formation, migration, and metastasis of A549 lung cancer cells.

Lactosylceramide-Induced Phosphorylation Signaling to Group IVA Phospholipase A2 via Reactive Oxygen Species in Tumor Necrosis Factor-α-Treated Cells.

The activity of α-type cytosolic phospholipase A2 (cPLA2 α, group IVA PLA2 ), which releases arachidonic acid (AA), is mainly regulated by the Ca2+ -induced intracellular translocation/attachment of the enzyme to substrate membranes and its phosphorylation. We previously reported that tumor necrosis factor-α (TNFα) stimulated the formation of lactosylceramide (LacCer) in L929 fibroblast cells, and this lipid directly bound with and activated cPLA2 α [Nakamura et al. [2013] J. Biol. Chem. 288:23264-23272]. We herein investigated the role of phosphorylation signaling in the TNFα/LacCer-induced activation of cPLA2 α in cells. TNFα-treated L929 cells released AA via the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and cPLA2 α, while a treatment with LacCer alone released AA in a similar manner. The TNFα-induced responses including release of AA were decreased by the inhibition of LacCer synthesis. The treatment with TNFα and LacCer increased the levels of reactive oxygen species (ROS), and the reduction/scavenging of ROS decreased the phosphorylation cascade and release of AA in TNFα/LacCer-treated L929 cells. In the cell line CHO, the treatment with LacCer stimulated the phosphorylation cascade and release of AA via the formation of ROS. Treatments with the anti-LacCer antibody and 4ß-phorbol 12-myristate 13-acetate stimulated the phosphorylation cascade, but did not release AA by itself. When combined with the Ca2+ ionophore A23187, treatments with the anti-LacCer antibody and 4ß-phorbol 12-myristate 13-acetate released AA. These results, including our previous findings, showed that LacCer alone simultaneously stimulates two processes to activate cPLA2 α: a phosphorylation signal and attachment of the enzyme to substrate membranes. J. Cell. Biochem. 118: 4370-4382, 2017. © 2017 Wiley Periodicals, Inc.

A common active site of polyhydroxyalkanoate synthase from Bacillus cereus YB-4 is involved in polymerization and alcoholysis reactions.

Polyhydroxyalkanoate (PHA) synthase from Bacillus cereus YB-4 (PhaRCYB4) catalyzes not only PHA polymerization but also alcoholytic cleavage of PHA chains. The alcoholysis activity of PhaRCYB4 is expressed when a hydroxyacyl-CoA monomer is absent but an alcohol compound is present. In this study, we performed alanine mutagenesis of the putative catalytic triad (Cys(151), Asp(306), and His(335)) in the PhaCYB4 subunit to identify the active site residues for polymerization and alcoholysis activities. Individual substitution of each triad residue with alanine resulted in loss of both polymerization and alcoholysis activities, suggesting that these residues are commonly shared between polymerization and alcoholysis reactions. The loss of activity was also observed following mutagenesis of the triad to other amino acids, except for one PhaRCYB4 mutant with a C151S substitution, which lost polymerization activity but still possessed cleavage activity towards PHA chains. The low-molecular-weight PHA isolated from the PhaRCYB4(C151S)-expressing strain showed a lower ratio of alcohol capping at the P(3HB) carboxy terminus than did that from the wild-type-expressing strain. This observation implies that hydrolysis activity of PhaRCYB4 might be elicited by the C151S mutation.

Alcoholytic cleavage of polyhydroxyalkanoate chains by class IV synthases induced by endogenous and exogenous ethanol.

Polyhydroxyalkanoate (PHA)-producing Bacillus strains express class IV PHA synthase, which is composed of the subunits PhaR and PhaC. Recombinant Escherichia coli expressing PHA synthase from Bacillus cereus strain YB-4 (PhaRCYB-4) showed an unusual reduction of the molecular weight of PHA produced during the stationary phase of growth. Nuclear magnetic resonance analysis of the low-molecular-weight PHA revealed that its carboxy end structure was capped by ethanol, suggesting that the molecular weight reduction was the result of alcoholytic cleavage of PHA chains by PhaRCYB-4 induced by endogenous ethanol. This scission reaction was also induced by exogenous ethanol in both in vivo and in vitro assays. In addition, PhaRCYB-4 was observed to have alcoholysis activity for PHA chains synthesized by other synthases. The PHA synthase from Bacillus megaterium (PhaRCBm) from another subgroup of class IV synthases was also assayed and was shown to have weak alcoholysis activity for PHA chains. These results suggest that class IV synthases may commonly share alcoholysis activity as an inherent feature.

Characterization of site-specific mutations in a short-chain-length/medium-chain-length polyhydroxyalkanoate synthase: in vivo and in vitro studies of enzymatic activity and substrate specificity.

Saturation point mutagenesis was carried out at position 479 in the polyhydroxyalkanoate (PHA) synthase from Chromobacterium sp. strain USM2 (PhaC(Cs)) with specificities for short-chain-length (SCL) [(R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyvalerate (3HV)] and medium-chain-length (MCL) [(R)-3-hydroxyhexanoate (3HHx)] monomers in an effort to enhance the specificity of the enzyme for 3HHx. A maximum 4-fold increase in 3HHx incorporation and a 1.6-fold increase in PHA biosynthesis, more than the wild-type synthase, was achieved using selected mutant synthases. These increases were subsequently correlated with improved synthase activity and increased preference of PhaC(Cs) for 3HHx monomers. We found that substitutions with uncharged residues were beneficial, as they resulted in enhanced PHA production and/or 3HHx incorporation. Further analysis led to postulations that the size and geometry of the substrate-binding pocket are determinants of PHA accumulation, 3HHx fraction, and chain length specificity. In vitro activities for polymerization of 3HV and 3HHx monomers were consistent with in vivo substrate specificities. Ultimately, the preference shown by wild-type and mutant synthases for either SCL (C(4) and C(5)) or MCL (C(6)) substrates substantiates the fundamental classification of PHA synthases.

In vitro evidence of chain transfer to tetraethylene glycols in enzymatic polymerization of polyhydroxyalkanoate.

A polyhydroxyalkanoate (PHA) was enzymatically synthesized in vitro, and the end structure of PHA associated with a chain transfer (CT) reaction was investigated. In the CT reaction, PHA chain transfers from PHA synthase (PhaC) to a CT agent, resulting in covalent bonding of CT agent to the PHA chain at its carboxyl end. In vitro CT reaction has never been demonstrated because of relatively low yields of in vitro synthesized poly[(R)-3-hydroxybutyrate)] (P(3HB)), which makes it difficult to characterize the end structures of the polymers by nuclear magnetic resonance (NMR). To overcome these difficulties, a novel in vitro synthesis method that produced relatively larger amounts of P(3HB) was developed by employing PhaCDa from Delftia acidovorans and two enantioselective enoyl-coenzyme A (CoA) hydratases which were R-hydratase (PhaJAc) from Aeromonas caviae and S-hydratase (FadB1x) from Pseudomonas putida KT2440 with ß-butyrolactone and CoA as starting materials. Using this method, P(3HB) synthesis was performed with tetraethylene glycols (TEGs) as a discriminable CT agent, and the resultant P(3HB) was characterized by (1)H-NMR. NMR analysis revealed that the carboxylic end of P(3HB) was covalently linked to TEGs, providing the first direct evidence of in vitro CT reaction.

Polyhydroxyalkanoate (PHA) synthesis by class IV PHA synthases employing Ralstonia eutropha PHB(-)4 as host strain.

Class IV polyhydroxyalkanoate (PHA) synthase from Bacillus cereus YB-4 (PhaRC(YB4)) or B. megaterium NBRC15308(T) (PhaRC(Bm)) was expressed in Ralstonia eutropha PHB(-)4 to compare the ability to produce PHA and the substrate specificity of PhaRCs. PhaRC(YB4) produced significant amounts of PHA and had broader substrate specificity than PhaRC(Bm).

Molecular weight change of polyhydroxyalkanoate (PHA) caused by the PhaC subunit of PHA synthase from Bacillus cereus YB-4 in recombinant Escherichia coli.

Polyhydroxyalkanoate (PHA)-producing Bacillus strains possess class IV PHA synthases composed of two subunit types, namely, PhaR and PhaC. In the present study, PHA synthases from Bacillus megaterium NBRC15308(T) (PhaRC(Bm)), B. cereus YB-4 (PhaRC(YB4)), and hybrids (PhaR(Bm)C(YB4) and PhaR(YB4)C(Bm)) were expressed in Escherichia coli JM109 to characterize the molecular weight of the synthesized poly(3-hydroxybutyrate) [P(3HB)]. PhaRC(Bm) synthesized P(3HB) with a relatively high molecular weight (M(n) = 890 × 10(3)) during 72 h of cultivation, whereas PhaRC(YB4) synthesized low-molecular-weight P(3HB) (M(n) = 20 × 10(3)). The molecular weight of P(3HB) synthesized by PhaRC(YB4) decreased with increasing culture time and temperature. This time-dependent behavior was observed for hybrid synthase PhaR(Bm)C(YB4), but not for PhaR(YB4)C(Bm). These results suggest that the molecular weight change is caused by the PhaC(YB4) subunit. The homology between PhaCs from B. megaterium and B. cereus YB-4 is 71% (amino acid identity); however, PhaC(YB4) was found to have a previously unknown effect on the molecular weight of the P(3HB) synthesized in E. coli.

Chain transfer reaction catalyzed by various polyhydroxyalkanoate synthases with poly(ethylene glycol) as an exogenous chain transfer agent.

Polyhydroxyalkanoate (PHA) synthases catalyze chain transfer (CT) reaction after polymerization reaction of PHA by transferring PHA chain from PHA synthase to a CT agent, resulting in covalent bonding of CT agent to PHA chain at the carboxyl end. Previous studies have shown that poly(ethylene glycol) (PEG) is an effective exogenous CT agent. This study aimed to compare the effects of PEG on CT reaction during poly[(R)-3-hydroxybutyrate] [P(3HB)] synthesis by using six PHA synthases in Escherichia coli JM109. The synthesized P(3HB) polymers were characterized in terms of molecular weight and end-group structure. Supplementation of PEG to the culture medium reduced P(3HB) molecular weights by up to 96% due to PEG-induced CT reaction. The P(3HB) polymers were subjected to (1)H NMR analysis to confirm the formation of a covalent bond between PEG and P(3HB) chain at the carboxyl end. This study revealed the reactivity of PHA synthases to PEG with respect to CT reaction in E. coli.