Identification of phosphorylation sites on ß1-adrenergic receptor in the mouse heart.

ß1-adrenergic receptor (Adrb1) belongs to the superfamily of G-protein-coupled receptors (GPCRs) and plays a critical role in the regulation of heart rate and myocardial contraction force. GPCRs are phosphorylated at multiple sites to regulate distinct signal transduction pathways in different tissues. However, little is known about the location and function of distinct phosphorylation sites of Adrb1 in vivo. To clarify the mechanisms underlying functional regulation associated with Adrb1 phosphorylation in vivo, we aimed to identify Adrb1 phosphorylation sites in the mouse heart using phosphoproteomics techniques with nano-flow liquid chromatography/tandem mass spectrometry (LC-MS/MS). We revealed the phosphorylation residues of Adrb1 to be Ser274 and Ser280 in the third intracellular loop and Ser412, Ser417, Ser450, Ser451, and Ser462 at the C-terminus. We also found that phosphorylation at Ser274, Ser280, and Ser462 was enhanced in response to stimulation with an Adrb1 agonist. This is the first study to identify Adrb1 phosphorylation sites in vivo. These findings will provide novel insights into the regulatory mechanisms mediated by Adrb1 phosphorylation.

Quantitative protein profiling of phenobarbital-induced drug metabolizing enzymes in rat liver by liquid chromatography mass spectrometry using formalin-fixed paraffin-embedded samples.

Administration of a compound can induce drug-metabolizing enzymes (DMEs) in the liver. DME induction can affect various parameters in toxicology studies. Therefore, evaluation of DME induction is important for interpreting test compound-induced biological responses. Several methods such as measurement of hepatic microsomal DME activity using substrates, electron microscopy, or immunohistochemistry have been used; however, these methods are limited in throughput and specificity or are not quantitative. Liquid chromatography mass spectrometry (LC/MS)-based protein analysis can detect and quantify multiple proteins simultaneously per assay. Studies have shown that formalin-fixed paraffin-embedded (FFPE) samples, which are routinely collected in toxicology studies, can be used for LC/MS-based protein analysis. To validate the utility of LC/MS using FFPE samples for quantitative evaluation of DME induction, we treated rats with a DME inducer, phenobarbital, and compared the protein expression levels of 13 phase-I and 11 phase-II DMEs between FFPE and fresh frozen hepatic samples using LC/MS. A good correlation between data from FFPE and frozen samples was obtained after analysis. In FFPE and frozen samples, the expression of 6 phase-I and 8 phase-II DMEs showed a similar significant increase and a prominent rise in Cyp2b2 and Cyp3a1 levels. In addition, LC/MS data were consistent with the measurement of microsomal DME activities. These results suggest that LC/MS-based protein expression analysis using FFPE samples is as effective as that using frozen samples for detecting DME induction.

A CDC7 inhibitor sensitizes DNA-damaging chemotherapies by suppressing homologous recombination repair to delay DNA damage recovery.

Cell division cycle 7 (CDC7), a serine/threonine kinase, plays important roles in DNA replication. We developed a highly specific CDC7 inhibitor, TAK-931, as a clinical cancer therapeutic agent. This study aimed to identify the potential combination partners of TAK-931 for guiding its clinical development strategies. Unbiased high-throughput chemical screening revealed that the highest synergistic antiproliferative effects observed were the combinations of DNA-damaging agents with TAK-931. Functional phosphoproteomic analysis demonstrated that TAK-931 suppressed homologous recombination repair activity, delayed recovery from double-strand breaks, and led to accumulation of DNA damages in the combination. Whole-genome small interfering RNA library screening identified sensitivity-modulating molecules, which propose the experimentally predicted target cancer types for the combination, including pancreatic, esophageal, ovarian, and breast cancers. The efficacy of combination therapy in these cancer types was preclinically confirmed in the corresponding primary-derived xenograft models. Thus, our findings would be helpful to guide the future clinical strategies for TAK-931.

Characterization of Postprandial Effects on CSF Metabolomics: A Pilot Study with Parallel Comparison to Plasma.

Cerebrospinal fluid (CSF) metabolites reflect biochemical diffusion/export from the brain and possibly serve as biomarkers related to brain disease severity, pathophysiology, and therapeutic efficacy/toxicity. Metabolomic studies using blood matrices have demonstrated interindividual and preanalytical variation of blood metabolites, whereas those of CSF metabolites remain unclear. In this study, we aimed to delineate the postprandial effects on CSF metabolites because fasting of patients with brain-related disorders is challenging. We collected pre- and postprandial (1.5, 3, and 6 h) plasma and CSF from nine healthy subjects. Using a mass-spectrometry-based global metabolomics approach, 150 and 130 hydrophilic metabolites and 263 and 340 lipids were detected in CSF and plasma, respectively. Principal component analysis of CSF hydrophilic metabolites and lipids primarily classified individual subjects at any time point, suggesting that the postprandial effects had a lower impact than interindividual variations on CSF metabolites. Individually, less than 10% of the CSF metabolites were putatively altered by postprandial effects (with either significant differences or over 2-fold changes, but not both) at any time point. Thus, global CSF metabolite levels are not directly associated with food intake, and except for several putatively altered CSF metabolites, postprandial effects are not a major concern when applying CSF metabolomics to screen biomarkers.

Molecular mechanism and potential target indication of TAK-931, a novel CDC7-selective inhibitor.

Replication stress (RS) is a cancer hallmark; chemotherapeutic drugs targeting RS are widely used as treatments for various cancers. To develop next-generation RS-inducing anticancer drugs, cell division cycle 7 (CDC7) has recently attracted attention as a target. We have developed an oral CDC7-selective inhibitor, TAK-931, as a candidate clinical anticancer drug. TAK-931 induced S phase delay and RS. TAK-931-induced RS caused mitotic aberrations through centrosome dysregulation and chromosome missegregation, resulting in irreversible antiproliferative effects in cancer cells. TAK-931 exhibited significant antiproliferative activity in preclinical animal models. Furthermore, in indication-seeking studies using large-scale cell panel data, TAK-931 exhibited higher antiproliferative activities in RAS-mutant versus RAS-wild-type cells; this finding was confirmed in pancreatic patient-derived xenografts. Comparison analysis of cell panel data also demonstrated a unique efficacy spectrum for TAK-931 compared with currently used chemotherapeutic drugs. Our findings help to elucidate the molecular mechanisms for TAK-931 and identify potential target indications.

Biologic Response of Colorectal Cancer Xenograft Tumors to Sequential Treatment with Panitumumab and Bevacizumab.

Recent studies in RAS wild-type (WT) metastatic colorectal cancer (mCRC) suggest that the survival benefits of therapy using anti-epidermal growth factor receptor (anti-EGFR) and anti-vascular endothelial growth factor (anti-VEGF) antibodies combined with chemotherapy are maximized when the anti-EGFR antibody is given as first-line, followed by subsequent anti-VEGF antibody therapy. We report reverse-translational research using LIM1215 xenografts of RAS WT mCRC to elucidate the biologic mechanisms underlying this clinical observation. Sequential administration of panitumumab then bevacizumab (PB) demonstrated a stronger tendency to inhibit tumor growth than bevacizumab then panitumumab (BP). Cell proliferation was reduced significantly with PB (P < .01) but not with BP based on Ki-67 index. Phosphoproteomic analysis demonstrated reduced phosphorylation of EGFR and EPHA2 with PB and BP compared with control. Western blotting showed reduced EPHA2 expression and S897-phosphorylation with PB; RSK phosphorylation was largely unaffected by PB but increased significantly with BP. In quantitative real-time PCR analyses, PB significantly reduced the expression of both lipogenic (FASN, MVD) and hypoxia-related (CA9, TGFBI) genes versus control. These results suggest that numerous mechanisms at the levels of gene expression, protein expression, and protein phosphorylation may explain the improved clinical activity of PB over BP in patients with RAS WT mCRC.

Panitumumab interaction with TAS-102 leads to combinational anticancer effects via blocking of EGFR-mediated tumor response to trifluridine.

Panitumumab is a monoclonal antibody developed against the human epidermal growth factor receptor (EGFR). TAS-102 is a novel chemotherapeutic agent containing trifluridine (FTD) as the active cytotoxic component. Both panitumumab and TAS-102 have been approved for the treatment of metastatic colorectal cancer. In this study, we revealed the mechanism underlying the anticancer effects of panitumumab/TAS-102 combination using preclinical models. Panitumumab/FTD cotreatment showed additive antiproliferative effects in LIM1215 and synergistic antiproliferative effects in SW48 colon cancer cells. Consistent with the in vitro effects, panitumumab/TAS-102 combination caused tumor regression in LIM1215 and COL-01-JCK colon cancer patient-derived xenograft models. In LIM1215 cells, FTD induced extracellular signal-regulated kinase (ERK)/protein kinase B (AKT)/signal transducer and activator of transcription 3 (STAT3) phosphorylation and subsequent serine/threonine phosphorylation of EGFR, while it had no effects on EGFR tyrosine phosphorylation. Panitumumab and the tyrosine kinase inhibitor erlotinib reduced the basal level of EGFR tyrosine phosphorylation and reversed FTD-induced ERK/AKT/STAT3 and EGFR serine/threonine phosphorylation. These results suggested that FTD in combination with the basal activity of EGFR tyrosine kinase induced downstream prosurvival signaling through ERK/AKT/STAT3 phosphorylation. Collectively, we propose that panitumumab interacts with FTD by targeting EGFR-mediated adaptive responses, thereby exerting anticancer effects when used in combination with TAS-102. These preclinical findings provide a compelling rationale for evaluating the combination of anti-EGFR antibodies with TAS-102 against metastatic colorectal cancer.

Strategy for Identification of Phosphorylation Levels of Low Abundance Proteins in Vivo for Which Antibodies Are not Available.

Protein function is mainly modulated by dynamic reversible or irreversible post-translational modifications. Among them, the identification of protein phosphorylation sites and changes in phosphorylation levels in vivo are of considerable interest for a better understanding of the protein function. Thus, effective strategies for the quantitative determination of phosphorylation degrees for low abundant proteins, for which antibodies are not available, are required in order to evaluate the functional regulation of proteins attributed to phosphorylation. In this study, we used the heart ß1-adrenergic receptor (Adrb1) as a model protein and developed FLAG-Adrb1 knock-in mice, in which the FLAG tag was inserted at the N-terminus of Adrb1. The phosphorylation sites and levels of Adrb1 in the heart were elucidated by immuno-affinity purification followed by quantitative mass spectrometry analysis using ion intensity ratio of the phosphorylated peptide versus corresponding unphosphorylated peptide. The phosphorylation levels at Ser274 and Ser462 of Adrb1 were approximately 0.25 and 0.0023. This effective strategy should be useful for not only analyzing site-specific phosphorylation levels of target proteins, but also quantifying the expression levels of proteins of interest when appropriate antibodies are not available.