Nucleos(t)ide analogs for hepatitis B virus infection differentially regulate the growth factor signaling in hepatocytes.

BACKGROUND: Recent clinical studies have suggested that the risk of developing HCC might be lower in patients with chronic hepatitis B receiving tenofovir disoproxil fumarate than in patients receiving entecavir, although there is no difference in biochemical and virological remission between the 2 drugs. METHODS: The effects of nucleoside analogs (NsAs; lamivudine and entecavir) or nucleotide analogs (NtAs; adefovir disoproxil, tenofovir disoproxil fumarate, and tenofovir alafenamide) on cell growth and the expression of growth signaling molecules in hepatoma cell lines and PXB cells were investigated in vitro. The tumor inhibitory effects of NsAs or NtAs were evaluated using a mouse xenograft model, and protein phosphorylation profiles were investigated. The binding of NsAs or NtAs to the insulin receptor (INSR) was investigated by thermal shift assays. RESULTS: NtAs, but not NsAs, showed direct growth inhibitory effects on hepatoma cell lines in vitro and a mouse model in vivo. A phosphoprotein array revealed that INSR signaling was impaired and the levels of phosphorylated (p)-INSRß and downstream molecules phosphorylated (p)-IRS1, p-AKT, p-Gab1, and p-SHP2 were substantially reduced by NtAs. In addition, p-epidermal growth factor receptor and p-AKT levels were substantially reduced by NtAs. Similar findings were also found in PXB cells and nontumor lesions of liver tissues from patients with chronic hepatitis B. Prodrug NtAs, but not their metabolites (adefovir, adefovir monophosphate, adefovir diphosphate, tenofovir, tenofovir monophosphate, and tenofovir diphosphate), had such effects. A thermal shift assay showed the binding of NtAs to INSRß. CONCLUSIONS: NtAs (adefovir disoproxil, tenofovir disoproxil fumarate, and tenofovir alafenamide), which are adenine derivative acyclic nucleotide analogs, potentially bind to the ATP-binding site of growth factor receptors and inhibit their autophosphorylation, which might reduce the risk of HCC in patients with chronic hepatitis B.

Lansoprazole inhibits the development of sessile serrated lesions by inducing G1 arrest via Skp2/p27 signaling pathway.

BACKGROUND: Although the serrated-neoplasia pathway reportedly accounts for 15-30% of colorectal cancer (CRC), no studies on chemoprevention of sessile serrated lesions (SSLs) have been reported. We searched for effective compounds comprehensively from a large series of compounds by employing Connectivity Map (CMAP) analysis of SSL-specific gene expression profiles coupled with in vitro screening using SSL patient-derived organoids (PDOs), and validated their efficacy using a xenograft mouse model of SSL. METHODS: We generated SSL-specific gene signatures based on DNA microarray data, and applied them to CMAP analysis with 1309 FDA-approved compounds to select candidate compounds. We evaluated their inhibitory effects on SSL-PDOs using a cell viability assay. SSL-PDOs were orthotopically transplanted into NOG mice for in vivo evaluation. The signal transduction pathway was evaluated by gene expression profile and protein expression analysis. RESULTS: We identified 221 compounds by employing CMAP analysis of SSL-specific signatures, which should cancel the gene signatures, and narrowed them down to 17 compounds. Cell viability assay using SSL-PDOs identified lansoprazole as having the lowest IC50 value (47 µM) among 17 compounds. When SSL-PDO was orthotopically transplanted into murine intestinal tract, the tumor grew gradually. Administration of lansoprazole to mice inhibited the growth of SSL xenograft whereas the tumor in control mice treated with vehicle alone grew gradually over time. The Ki67 index in xenograft lesions from the lansoprazole group was significantly lower compared with the control group. Cell cycle analysis of SSL-PDOs treated with lansoprazole exhibited a significant increase in G1 phase cell population. Microarray and protein analysis revealed that lansoprazole downregulated Skp2 expression and upregulated p27 expression in SSL-PDOs. CONCLUSIONS: Our data strongly suggest that lansoprazole is the most effective chemopreventive agent against SSL, and that lansoprazole induces G1 cell cycle arrest by downregulating Skp2 and upregulating p27 in SSL cells.

Synthesis and Bioevaluation of 3-(Arylmethylene)indole Derivatives: Discovery of a Novel ALK Modulator with Antiglioblastoma Activities.

Glioblastoma is the most common brain tumor, with high recurrence and low survival rates. An integrative bioinformatics analysis demonstrated that anaplastic lymphoma kinase (ALK) is a promising therapeutic target for glioblastoma. We designed and synthesized a series of 3-(arylmethylene)indole derivatives, which were further evaluated for antiproliferative activity using glioma cell lines. Among them, compound 4a significantly inhibited the viability of glioblastoma cells. With favorable pharmacokinetic characteristics and blood-brain barrier permeability, 4a improved the survival rate and inhibited the growth of orthotopic glioblastoma. The Phospho-Totum system revealed that ALK was a potential target for the antiglioblastoma activity of 4a. Further experiments indicated that 4a might be a novel ALK modulator, which interacted with the extracellular ligand-binding domain of ALK, thus selectively induced ERK-mediated autophagy and apoptosis. Our findings provide an alternative ALK-based targeting strategy and a new drug candidate for glioblastoma therapy.

A Th1-like CD4+ T-cell Cluster That Predicts Disease-free Survival in Early-stage Lung Cancer.

Perioperative immune checkpoint inhibitors have been shown to improve prognosis in early-stage lung cancer. However, no biomarkers are known to indicate the requirement for treatment. This study aimed to identify T-cell clusters responsible for antitumor immunity in patients with early-stage lung cancer. Preoperative blood samples from 50 consecutive patients with lung cancer who were diagnosed as operable and underwent complete resection were analyzed by mass cytometry. Patients were divided into two groups: no recurrence at a minimum observation period of 851 days (median observation period: 1,031.5 days) and recurrence by the last observation date. Mass cytometry and single-cell RNA sequencing analysis of lymph nodes (LN) and tumor-infiltrating T cells were also performed. CCR4-CCR6+ Th7R showed discriminative ability between recurrence and non-recurrence patients with lung cancer. Patients with more than 3.04% Th7R showed significantly favorable disease-free survival. Th7R was a major component of CD4+ T cells in tumor microenvironments and LNs adjacent to lung cancer tissues and was the only cluster that decreased in peripheral blood after the removal of cancer tissues, suggesting that Th7R was primed and proliferated in tumor-draining LNs in the presence of cancer tissues. Th7R had the kinetics that antitumor T cells should have, as indicated by the cancer immunity cycle; thus, peripheral blood Th7R could represent the potency of tumor immunity by reflecting priming and proliferation in tumor-draining LNs and Th7R in the tumor microenvironment. Prediction using peripheral Th7R before surgery could allow the selection of patients who require perioperative drug therapy and optimize therapeutic interventions with clinical implications. Significance: Peripheral Th7R, a Th1-like CD4+ T-cell cluster reflecting priming status in draining LNs and immune status in the tumor microenvironment, predicts disease-free survival after complete resection and has significant clinical relevance in selecting appropriate therapeutic interventions in patients with early-stage lung cancer.

Single-Cell Analysis Reveals a CD4+ T-cell Cluster That Correlates with PD-1 Blockade Efficacy.

CD4+ T-cell immunity helps clonal proliferation, migration, and cancer cell killing activity of CD8+ T cells and is essential in antitumor immune responses. To identify CD4+ T-cell clusters responsible for antitumor immunity, we simultaneously analyzed the naïve-effector state, Th polarization, and T-cell receptor clonotype based on single-cell RNA-sequencing data. Unsupervised clustering analysis uncovered the presence of a new CD4+ T-cell metacluster in the CD62Llow CD4+ T-cell subpopulation, which contained multicellular clonotypes associated with efficacy of programmed death-ligand 1 (PD-1) blockade therapy. The CD4+ T-cell metacluster consisted of CXCR3+CCR4-CCR6+ and CXCR3-CCR4-CCR6+ cells and was characterized by high expression of IL7 receptor and TCF7. The frequency of these cells in the peripheral blood significantly correlated with progression-free survival and overall survival of patients with lung cancer after PD-1 blockade therapy. In addition, the CD4+ metacluster in the peripheral blood correlated with CD4+ T-cell infiltration in the tumor microenvironment, whereas peripheral Th1 correlated with local CD8+ T-cell infiltration. Together, these findings suggest that CD62Llow CCR4-CCR6+ CD4+ T cells form a novel metacluster with predictive potential of the immune status and sensitivity to PD-1 blockade, which may pave the way for personalized antitumor immunotherapy strategies for patients. SIGNIFICANCE: The identification of a new CD4+ T-cell metacluster that corresponds with immune status could guide effective tumor treatment by predicting response to immunotherapy using peripheral blood samples from patients.

Resveratrol inhibits development of colorectal adenoma via suppression of LEF1; comprehensive analysis with connectivity map.

Although many chemopreventive studies on colorectal tumors have been reported, no effective and safe preventive agent is currently available. We searched for candidate preventive compounds against colorectal tumor comprehensively from United States Food and Drug Administration (FDA)-approved compounds by using connectivity map (CMAP) analysis coupled with in vitro screening with colorectal adenoma (CRA) patient-derived organoids (PDOs). We generated CRA-specific gene signatures based on the DNA microarray analysis of CRA and normal epithelial specimens, applied them to CMAP analysis with 1309 FDA-approved compounds, and identified 121 candidate compounds that should cancel the gene signatures. We narrowed them down to 15 compounds, and evaluated their inhibitory effects on the growth of CRA-PDOs in vitro. We finally identified resveratrol, one of the polyphenolic phytochemicals, as a compound showing the strongest inhibitory effect on the growth of CRA-PDOs compared with normal epithelial PDOs. When resveratrol was administered to ApcMin/+ mice at 15 or 30 mg/kg, the number of polyps (adenomas) was significantly reduced in both groups compared with control mice. Similarly, the number of polyps (adenomas) was significantly reduced in azoxymethane-injected rats treated with 10 or 100 mg/resveratrol compared with control rats. Microarray analysis of adenomas from resveratrol-treated rats revealed the highest change (downregulation) in expression of LEF1, a key molecule in the Wnt signaling pathway. Treatment with resveratrol significantly downregulated the Wnt-target gene (MYC) in CRA-PDOs. Our data demonstrated that resveratrol can be the most effective compound for chemoprevention of colorectal tumors, the efficacy of which is mediated through suppression of LEF1 expression in the Wnt signaling pathway.

Phosprof: pathway analysis database of drug response based on phosphorylation activity measurements.

Protein phosphorylation plays a fundamental role in many cellular processes. Proteins are phosphorylated by kinases, which have been studied as drug targets for the treatment of various diseases, particularly cancer. Because kinases have multiple roles in interconnected molecular pathways, their specific regulation is required to enhance beneficial and reduce adversarial effects of drugs. Using our previously developed platform, we measured phosphorylation profiles of MCF7 and K562 cells treated with 94 clinical drugs. These phosphorylation profiles can provide insights into pathway activities and biological functions. Here, we introduce Phosprof, a novel database of drug response based on phosphorylation activity. Phosprof is able to present up- or downregulated phosphorylated signature proteins on pathway maps, significant pathways on the hierarchal tree in signal transduction and commonly perturbed pathways affected by the selected drugs. It also serves as a useful web interface for new or known drug profile search based on their molecular similarity with the 94 drugs. Phosprof can be helpful for further investigation of drug responses in terms of phosphorylation by utilizing the various approved drugs whose target phenotypes are known. DATABASE URL: https://phosprof.medals.jp/.

Medical database analysis of japanese multiple myeloma patients with planned stem cell transplantation (MEDALIST) - a focus on healthcare resource utilization and cost.

This study explored the burden associated with stem cell mobilization, with or without cyclophosphamide (CPA), in patients who intended to receive autologous stem cell transplantation (ASCT) for multiple myeloma (MM). A Japanese health care claims database (MDV) was used to analyze the health care resource utilization patterns and medical cost between 2013 and 2016 (pre-plerixafor launch). The patients were further categorized into groups who received granulocyte-colony stimulating factor (G-CSF) alone or G-CSF + CPA group and analyzed in both mobilization and ASCT phases of treatment. Overall, there were more MM patients who were treated with G-CSF + CPA combination therapy than G-CSF alone. Length-of-stay was 1.6 times longer in the combination group during the mobilization phase. A reverse trend was observed during the ASCT phase. Direct cost was approximately 1.2 million yen during the mobilization phase and 2.3 million yen during the ASCT phase, with hospitalization basic fee accounting for the highest proportion in both groups and phases. A substantial amount of healthcare resource and cost was consumed in both phases. This study may serve as a basic reference for further health technology assessment of new medicines such as plerixafor. Further investigation of differences between treatment groups is warranted.

Potential use of lenvatinib for patients with unresectable hepatocellular carcinoma including after treatment with sorafenib: Real-world evidence and in vitro assessment via protein phosphorylation array.

The efficacy and safety of lenvatinib (LEN) as a second/third-line treatment for unresectable hepatocellular carcinoma (HCC) after sorafenib (SOR) therapy remains unknown. We evaluated the outcomes of second/third-line LEN treatment, investigated the sensitivity of a SOR-resistant HCC cell line (PLC/PRF5-R2) to LEN, and assessed their signal transduction pathways by protein array analysis. We retrospectively enrolled 57 patients with unresectable HCC. Fifty-three radiologically evaluated patients comprised 34 molecular-targeted agent (MTA)-naive (first-line), nine intolerant to SOR (second-line), and 10 resistant to regorafenib (third-line). The objective response rates (ORRs) were 61.8% in first-line, 33.3% in second-line, and 20.0% in third-line groups. The overall survival (OS) in the first-line was significantly longer than that in the third-line group (p < 0.05). Patients with better liver functional reserves (child score, ALBI grade) exhibited higher ORR and longer OS. The IC50 of LEN against PLC/PRF5-R2 was significantly higher than that against PLC/PRF5. LEN significantly inhibited more LEN-related signal transduction pathways in PLC/PRF5 than in PLC/PRF5-R2 cells. This suggests that LEN is active and safe as a second/third-line treatment for unresectable HCC. LEN seems more effective for patients with HCC with better hepatic reserve functions or before MTA-resistance is acquired because of the partial cross-resistance to SOR.

Erratum: Author Correction: "Dysfunctions" induced by Roux-en-Y gastric bypass surgery are concomitant with metabolic improvement independent of weight loss.

[This corrects the article DOI: 10.1038/s41421-019-0138-2.].

"Dysfunctions" induced by Roux-en-Y gastric bypass surgery are concomitant with metabolic improvement independent of weight loss.

Metabolic surgery has been increasingly recommended for obese diabetic patients, but questions remain as to its molecular mechanism that leads to improved metabolic parameters independently of weight loss from a network viewpoint. We evaluated the role of the Roux limb (RL) in Roux-en-Y gastric bypass (RYGB) surgery in nonobese diabetic rat models. Improvements in metabolic parameters were greater in the long-RL RYGB group. Transcriptome profiles reveal that amelioration of diabetes state following RYGB differs remarkably from both normal and diabetic states. According to functional analysis, RYGB surgery significantly affected a major gene group, i.e., the newly changed group, which represented diabetes-irrelevant genes abnormally expressed after RYGB. We hypothesize that novel "dysfunctions" carried by this newly changed gene group induced by RYGB rebalance diabetic states and contribute to amelioration of metabolic parameters. An unusual increase in cholesterol (CHOL) biosynthesis in RL enriched by the newly changed group was concomitant with ameliorated metabolic parameters, as demonstrated by measurements of physiological parameters and biodistribution analysis using [14C]-labeled glucose. Our findings demonstrate RYGB-induced "dysfunctions" in the newly changed group as a compensatory role contributes to amelioration of diabetes. Rather than attempting to normalize "abnormal" molecules, we suggest a new disease treatment strategy of turning "normal" molecules "abnormal" in order to achieve a new "normal" physiological balance. It further implies a novel strategy for drug discovery, i.e. targeting also on "normal" molecules, which are traditionally ignored in pharmaceutical development.

IGF2 Autocrine-Mediated IGF1R Activation Is a Clinically Relevant Mechanism of Osimertinib Resistance in Lung Cancer.

EGFR-mutated lung cancer accounts for a significant proportion of lung cancer cases worldwide. For these cases, osimertinib, a third-generation EGFR tyrosine kinase inhibitor, is extensively used as a first-line or second-line treatment. However, lung cancer cells acquire resistance to osimertinib in 1 to 2 years. Thus, a thorough clarification of resistance mechanisms to osimertinib is highly anticipated. Recent next-generation sequencing (NGS) of lung cancer samples identified several genetically defined resistance mechanisms to osimertinib, such as EGFR C797S or MET amplification. However, nongenetically defined mechanisms are not well evaluated. For a thorough clarification of osimertinib resistance, both genetic and nongenetic mechanisms are essential. By using our comprehensive protein phosphorylation array, we detected IGF1R bypass pathway activation after EGFR abolishment. Both of our established lung cancer cells and patient-derived lung cancer cells demonstrated IGF2 autocrine-mediated IGF1R pathway activation as a mechanism of osimertinib resistance. Notably, this resistance mechanism was not detected by a previously performed NGS, highlighting the essential roles of living cancer cells for a thorough clarification of resistance mechanisms. Interestingly, the immunohistochemical analysis confirmed the increased IGF2 expression in lung cancer patients who were treated with osimertinib and met the established clinical definition of acquired resistance. The findings highlight the crucial roles of cell-autonomous ligand expression in osimertinib resistance. Here, we report for the first time the IGF2 autocrine-mediated IGF1R activation as a nongenetic mechanism of osimertinib resistance in lung cancer at a clinically relevant level. IMPLICATIONS: Using comprehensive protein phosphorylation array and patient-derived lung cancer cells, we found that IGF2 autocrine-mediated IGF1R pathway activation is a clinically relevant and common mechanism of acquired resistance to osimertinib.

Promotion of the Warburg effect is associated with poor benefit from adjuvant chemotherapy in colorectal cancer.

Metabolic reprogramming, including the Warburg effect, is a hallmark of cancer. Indeed, the diversity of cancer metabolism leads to cancer heterogeneity, but accurate assessment of metabolic properties in tumors has not yet been undertaken. Here, we performed absolute quantification of the expression levels of 113 proteins related to carbohydrate metabolism and antioxidant pathways, in stage III colorectal cancer surgical specimens from 70 patients. The Warburg effect appeared in absolute protein levels between tumor and normal mucosa specimens demonstrated. Notably, the levels of proteins associated with the tricarboxylic citric acid cycle were remarkably reduced in the malignant tumors which had relapsed after surgery and treatment with 5-fluorouracil-based adjuvant therapy. In addition, the efficacy of 5-fluorouracil also decreased in the cultured cancer cell lines with promotion of the Warburg effect. We further identified nine and eight important proteins, which are closely related to the Warburg effect, for relapse risk and 5-fluorouracil benefit, respectively, using a biomarker exploration procedure. These results provide us a clue for bridging between metabolic protein expression profiles and benefit from 5-fluorouracil adjuvant chemotherapy.

CD4+ T-cell Immunity in the Peripheral Blood Correlates with Response to Anti-PD-1 Therapy.

Accumulating evidence indicates that CD8+ T cells in the tumor microenvironment and systemic CD4+ T-cell immunity play an important role in mediating durable antitumor responses. We longitudinally examined T-cell immunity in the peripheral blood of patients with non-small lung cancer and found that responders had significantly (P < 0.0001) higher percentages of effector, CD62Llow CD4+ T cells prior to PD-1 blockade. Conversely, the percentage of CD25+FOXP3+ CD4+ T cells was significantly (P = 0.034) higher in nonresponders. We developed a formula, which demonstrated 85.7% sensitivity and 100% specificity, based on the percentages of CD62Llow CD4+ T cells and CD25+FOXP3+ cells to predict nonresponders. Mass cytometry analysis revealed that the CD62Llow CD4+ T-cell subset expressed T-bet+, CD27-, FOXP3-, and CXCR3+, indicative of a Th1 subpopulation. CD62Llow CD4+ T cells significantly correlated with effector CD8+ T cells (P = 0.0091) and with PD-1 expression on effector CD8+ T cells (P = 0.0015). Gene expression analysis revealed that CCL19, CLEC-2A, IFNA, IL7, TGFBR3, CXCR3, and HDAC9 were preferentially expressed in CD62Llow CD4+ T cells derived from responders. Notably, long-term responders, who had >500-day progression-free survival, showed significantly higher numbers of CD62Llow CD4+ T cells prior to PD-1 blockade therapy. Decreased CD62Llow CD4+ T-cell percentages after therapy resulted in acquired resistance, with long-term survivors maintaining high CD62Llow CD4+ T-cell percentages. These results pave the way for new treatment strategies for patients by monitoring CD4+ T-cell immune statuses in their peripheral blood.

ToGo-WF: prediction of RNA tertiary structures and RNA-RNA/protein interactions using the KNIME workflow.

Recent progress in molecular biology has revealed that many non-coding RNAs regulate gene expression or catalyze biochemical reactions in tumors, viruses and several other diseases. The tertiary structure of RNA molecules and RNA-RNA/protein interaction sites are of increasing importance as potential targets for new medicines that treat a broad array of human diseases. Current RNA drugs are split into two groups: antisense RNA molecules and aptamers. In this report, we present a novel workflow to predict RNA tertiary structures and RNA-RNA/protein interactions using the KNIME environment, which enabled us to assemble a combination of RNA-related analytical tools and databases. In this study, three analytical workflows for comprehensive structural analysis of RNA are introduced: (1) prediction of the tertiary structure of RNA; (2) prediction of the structure of RNA-RNA complexes and analysis of their interactions; and (3) prediction of the structure of RNA-protein complexes and analysis of their interactions. In an RNA-protein case study, we modeled the tertiary structure of pegaptanib, an aptamer drug, and performed docking calculations of the pegaptanib-vascular endothelial growth factor complex using a fragment of the interaction site of the aptamer. We also present molecular dynamics simulations of the RNA-protein complex to evaluate the affinity of the complex by mutating bases at the interaction interface. The results provide valuable information for designing novel features of aptamer-protein complexes.

Prospective exosome-focused translational research for afatinib study of non-small cell lung cancer patients expressing EGFR (EXTRA study).

Patients with EGFR-mutated non-small cell lung cancer (NSCLC) exhibit resistance to EGFR-tyrosine kinase inhibitors (TKIs) within 9-14 months of therapy. Recently, EGFR-mutated NSCLC has demonstrated the potential for heterogeneity; therefore, the manner of clonal heterogeneity may impact the duration of progression-free and overall survival and other parameters affecting EGFR-TKI treatment efficacy. However no predictive biomarker of these favorable treatment efficacies has been identified to date. The exosome-focused translational research for afatinib (EXTRA) study aims to identify a novel predictive biomarker and a resistance marker for afatinib by analyzing data from association studies of the clinical efficacy of afatinib and four "OMICs" (genomics, proteomics, epigenomics, and metabolomics) using peripheral blood from patients treated with afatinib. This study aims to: (i) conduct comprehensive multi-OMIC analyses in a prospective clinical trial, and (ii) focus on both sera/plasma and exosome as a source for OMIC analyses to identify a novel predictor of the efficacy of a specific drug. To eliminate the carryover bias of prior treatment, systemic treatment-naïve patients were enrolled. The candidates to be screened for biomarkers comprise a discovery cohort of 60 patients and an independent validation cohort of 40 patients. The EXTRA study is the first trial to screen novel biomarkers of longer treatment efficacy of EGFR-TKIs using four-OMICs analyses, focusing on both "naked or free" molecules and "capsulated" exosomal components in serially collected peripheral blood.

Plasma amino acid profile associated with fatty liver disease and co-occurrence of metabolic risk factors.

Fatty liver disease (FLD) increases the risk of diabetes, cardiovascular disease, and steatohepatitis, which leads to fibrosis, cirrhosis, and hepatocellular carcinoma. Thus, the early detection of FLD is necessary. We aimed to find a quantitative and feasible model for discriminating the FLD, based on plasma free amino acid (PFAA) profiles. We constructed models of the relationship between PFAA levels in 2,000 generally healthy Japanese subjects and the diagnosis of FLD by abdominal ultrasound scan by multiple logistic regression analysis with variable selection. The performance of these models for FLD discrimination was validated using an independent data set of 2,160 subjects. The generated PFAA-based model was able to identify FLD patients. The area under the receiver operating characteristic curve for the model was 0.83, which was higher than those of other existing liver function-associated markers ranging from 0.53 to 0.80. The value of the linear discriminant in the model yielded the adjusted odds ratio (with 95% confidence intervals) for a 1 standard deviation increase of 2.63 (2.14-3.25) in the multiple logistic regression analysis with known liver function-associated covariates. Interestingly, the linear discriminant values were significantly associated with the progression of FLD, and patients with nonalcoholic steatohepatitis also exhibited higher values.

Relationship between regulatory pattern of gene expression level and gene function.

Regulation of gene expression levels is essential for all living systems and transcription factors (TFs) are the main regulators of gene expression through their ability to repress or induce transcription. A balance between synthesis and degradation rates controls gene expression levels. To determine which rate is dominant, we analyzed the correlation between expression levels of a TF and its regulated gene based on a mathematical model. We selected about 280,000 expression patterns of 355 TFs and 647 regulated genes using DNA microarray data from the Gene Expression Omnibus (GEO) data repository. Based on our model, correlation between the expressions of TF-regulated gene pairs corresponds to tuning of the synthesis rate, whereas no correlation indicates excessive synthesis and requires tuning of the degradation rate. The gene expression relationships between TF-regulated gene pairs were classified into four types that correspond to different gene regulatory mechanisms. It was surprising that fewer than 20% of these genes were governed by the familiar regulatory mechanism, i.e., through the synthesis rate. Moreover, we performed pathway analysis and found that each classification type corresponded to distinct gene functions: cellular regulation pathways were dominant in the type with synthesis rate regulation and terms associated with diseases such as cancer, Parkinson's disease, and Alzheimer's disease were dominant in the type with degradation rate regulation. Interestingly, these diseases are caused by the accumulation of proteins. These results indicated that gene expression is regulated structurally, not arbitrarily, according to the gene function. This funding is indicative of a systematic control of transcription processes at the whole-cell level.

A large-scale targeted proteomics assay resource based on an in vitro human proteome.

Targeted proteomics approaches are of value for deep and accurate quantification of protein abundance. Extending such methods to quantify large numbers of proteins requires the construction of predefined targeted assays. We developed a targeted proteomics platform-in vitro proteome-assisted multiple reaction monitoring (MRM) for protein absolute quantification (iMPAQT)-by using >18,000 human recombinant proteins, thus enabling protein absolute quantification on a genome-wide scale. Our platform comprises experimentally confirmed MRM assays of mass tag (mTRAQ)-labeled peptides to allow for rapid and straightforward measurement of the absolute abundance of predefined sets of proteins by mass spectrometry. We applied iMPAQT to delineate the quantitative metabolic landscape of normal and transformed human fibroblasts. Oncogenic transformation gave rise to relatively small but global changes in metabolic pathways resulting in aerobic glycolysis (Warburg effect) and increased rates of macromolecule synthesis. iMPAQT should facilitate quantitative biology studies based on protein abundance measurements.

Cytokinetic Failure-induced Tetraploidy Develops into Aneuploidy, Triggering Skin Aging in Phosphovimentin-deficient Mice.

Tetraploidy, a state in which cells have doubled chromosomal sets, is observed in ∼20% of solid tumors and is considered to frequently precede aneuploidy in carcinogenesis. Tetraploidy is also detected during terminal differentiation and represents a hallmark of aging. Most tetraploid cultured cells are arrested by p53 stabilization. However, the fate of tetraploid cells in vivo remains largely unknown. Here, we analyze the ability to repair wounds in the skin of phosphovimentin-deficient (VIM(SA/SA)) mice. Early into wound healing, subcutaneous fibroblasts failed to undergo cytokinesis, resulting in binucleate tetraploidy. Accordingly, the mRNA level of p21 (a p53-responsive gene) was elevated in a VIM(SA/SA)-specific manner. Disappearance of tetraploidy coincided with an increase in aneuploidy. Thereafter, senescence-related markers were significantly elevated in VIM(SA/SA) mice. Because our tetraploidy-prone mouse model also exhibited subcutaneous fat loss at the age of 14 months, another premature aging phenotype, our data suggest that following cytokinetic failure, a subset of tetraploid cells enters a new cell cycle and develops into aneuploid cells in vivo, which promote premature aging.