Impact of different fibrin glue application methods on inguinal hernia mesh fixation capability.

The use of fibrin glue for inguinal hernia mesh fixation has been suggested to be effective in preventing hematomas and reducing postoperative pain compared to tacks and sutures.. The effect of fibrin glue can vary significantly based on the device used. This study assessed the efficacy of fibrin glue based on the type of devices used in an ex vivo system. The rabbit's abdominal wall was trimmed to a size of 3.0 × 6.0 cm and was secured at the edges with metal fixtures. To measure the maximum tensile strength at the point of adhesion failure, the hernia mesh was fixed to the rabbit's abdominal wall using fibrin glue in a 2 cm square area, left for 3 min, and then pulled at a speed of 50 cm/min. The test was conducted 10 times for each group. The median (minimum-maximum) tensile strength values using the spraying, two-liquid mixing, and sequential layering methods were 3.58 (1.99-4.95), 0.51 (0.27-1.89), and 1.32 (0.63-1.66) N, respectively. The spraying method had predominantly higher tensile strength values than the two-liquid mixing and sequential layering methods (P < 0.01). In conclusion, in hernia mesh fixation, the spraying method can be adopted to achieve appropriate adhesive effects.

Baseline ctDNA gene alterations as a biomarker of survival after panitumumab and chemotherapy in metastatic colorectal cancer.

Certain genetic alterations and right-sided primary tumor location are associated with resistance to anti-epidermal growth factor (EGFR) treatment in metastatic colorectal cancer (mCRC). The phase 3 PARADIGM trial (n = 802) demonstrated longer overall survival with first-line anti-EGFR (panitumumab) versus antivascular endothelial growth factor (bevacizumab) plus modified FOLFOX6 in patients with RAS wild-type mCRC with left-sided primary tumors. This prespecified exploratory biomarker analysis of PARADIGM (n = 733) evaluated the association between circulating tumor DNA (ctDNA) gene alterations and efficacy outcomes, focusing on a broad panel of gene alterations associated with resistance to EGFR inhibition, including KRAS, NRAS, PTEN and extracellular domain EGFR mutations, HER2 and MET amplifications, and ALK, RET and NTRK1 fusions. Overall survival was prolonged with panitumumab plus modified FOLFOX6 versus bevacizumab plus modified FOLFOX6 in patients with ctDNA that lacked gene alterations in the panel (that is, negative hyperselected; median in the overall population: 40.7 versus 34.4 months; hazard ratio, 0.76; 95% confidence interval, 0.62-0.92) but was similar or inferior with panitumumab in patients with ctDNA that contained any gene alteration in the panel (19.2 versus 22.2 months; hazard ratio, 1.13; 95% confidence interval, 0.83-1.53), regardless of tumor sidedness. Negative hyperselection using ctDNA may guide optimal treatment selection in patients with mCRC. ClinicalTrials.gov registrations: NCT02394834 and NCT02394795 .

A Direct Aspiration First Pass Technique for Basilar Artery Occlusion Caused by Elastic-hard Tumor Embolus via the Pulmonary Vein by Metastatic Prostate Adenocarcinoma: A Case Report.

Basilar artery occlusion (BAO) accounts for only 1% of all strokes, and cerebral infarction resulting from tumor emboli has been infrequently demonstrated; therefore, few reports described BAO due to tumor embolus and its treatment experience. We report here an 83-year-old man with an acute BAO caused by embolized lung tumor invading right pulmonary vein that was revealed as metastasis of prostate adenocarcinoma. The patient underwent rapid recanalization through acute thrombectomy with a direct aspiration first pass technique (ADAPT) with Penumbra catheter. Successful recanalization was achieved in reperfusion grade of thrombolysis in cerebral infarction (TICI) 2b, and the embolus revealed a highly elastic hard tumorous mass of which texture was too tough to be caught by stent retriever. Immunohistopathologic examination of the embolus revealed adenocaricinoma of the prostate. In spite of that the recanalization was obtained, the patient died of the brain stem infarction after 7 days from the onset. We experienced a rare case of acute BAO caused by embolized prostate cancer metastasizing lung and invading pulmonary vein. When we face to patients with lung tumor invading pulmonary vein, tumor embolus should have been strongly considered and aspiration thrombectomy may be safer and more effective for the condition because of the difficulty of predicting an embolus's texture before treatment.

Efficacy of the Envelope Method in Applying Polyglycolic Acid Sheets to Post-Endoscopic Submucosal Dissection Ulcers in Living Pigs.

BACKGROUND/AIMS: Application of polyglycolic acid (PGA) sheets using fibrin glue in post-endoscopic submucosal dissection (ESD) ulcers to prevent bleeding has been reported to be difficult with the conventional delivery method because of gravity. This study assessed the usefulness of the envelope-based delivery system with and against gravity in living pigs. METHODS: PGA sheets were applied on post-ESD ulcers with and against gravity six times each using the conventional and envelope methods, respectively. The PGA sheet delivery time and the endoscopic and histological findings of the treated ulcer floors were compared. RESULTS: With gravity, the median PGA sheet application time was 1.00 (0.68-1.30) min/cm2 and 0.32 (0.18-0.52) min/cm2 with the conventional and envelope techniques (p=0.002), respectively, and against gravity, it was 1.20 (1.13-1.63) min/cm2 and 0.50 (0.39-0.58) min/cm2 (p=0.002), respectively. Against gravity, the endoscopic and histological findings revealed that the conventional group had insufficient fixation of the PGA sheets, but the envelope groups had sufficient fixation. The results with gravity were similar between the groups. CONCLUSION: The envelope method makes it possible to deliver PGA sheets to the stomach quickly and cover ulcers appropriately both with and against gravity in living pigs.

Video-based assessment of drug-induced effects on contractile motion properties using human induced pluripotent stem cell-derived cardiomyocytes.

INTRODUCTION: Drug-induced inotropic change is a risk factor in drug development; thus, de-risking is desired in the early stages of drug development. Unlike proarrhythmic risk assessment using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), few in vitro models were validated to predict cardiac contractility. Motion field imaging (MFI), a high-resolution block matching-based optical flow technique, was expected to possess high quantitative predictivity in the detection of contraction speed. We aimed to establish an in vitro model to assess drug-induced contractile changes using hiPSC-CMs and MFI. METHODS: MFI was designed to noninvasively characterize cardiomyocyte contractile behavior by analyzing light microscope video images, and maximum contraction speed (MCS) was used as the index of contractility. Using MFI, 9 inactive compounds, 10 negative inotropes, and 10 positive inotropes were tested. Two negative chronotropes, ZD7288 and ivabradine, were also tested. To determine the sensitivity and specificity of the assay, the minimum effective concentration of the MCS was compared with the human effective total therapeutic concentration for 28 compounds in clinical use. RESULTS: For 8 negative and 8 positive inotropes, the effects observed in in vivo and clinical studies were detected in MFI assay. MFI assay showed negative chronotropic changes without inotropic changes. MFI assay presented sufficient specificity (83%) and sensitivity (88%), and RNA-sequence analysis provided an insight into the relationship between occurrence of the false compounds and target gene expression. DISCUSSION: We demonstrated the utility of MFI assay using hiPSC-CMs to assess drug-induced contractile function. These results will facilitate the de-risking of compounds during early drug development.

High-Throughput Screening to Evaluate Inhibition of Bile Acid Transporters Using Human Hepatocytes Isolated From Chimeric Mice.

Cholestasis resulting from hepatic bile acid efflux transporter inhibition may contribute to drug-induced liver injury (DILI). This condition is a common safety-related reason for drug attrition and withdrawal. To screen for safety risks associated with efflux transport inhibition, we developed a high-throughput cellular assay for different drug discovery phases. Hepatocytes isolated from chimeric mice with humanized livers presented gene expression resembling that of the human liver and demonstrated apical membrane polarity when sandwiched between Matrigel and collagen. The fluorescent bile acid-derivative cholyl-l-lysyl-fluorescein (CLF) was used to quantify drug-induced efflux transport inhibition in hepatocytes. Cyclosporine inhibited CLF accumulation in the apical bile canalicular lumen in a concentration-dependent manner. The assay had equivalent predictive power to a primary human hepatocyte-based assay and greater predictive power than an assay performed with rat hepatocytes. Predictive power was tested using 45 pharmaceutical compounds, and 91.3% of the compounds with cholestatic potential (21/23) had margins (IC50/Cmax) < 20. In contrast, 90.9% (20/22) of compounds without cholestatic potential had IC50/Cmax>20. Assay sensitivity and specificity were 91.3% and 90.9%, respectively. We suggest that this improved assay performance could result from higher expression of efflux transporters, metabolic pathways, and/or species differences. Given the long-term supply of cells from the same donor, the humanized mouse-derived hepatocyte-based CLF efflux assay could be a valuable tool for predicting cholestatic DILI.

Cell-based two-dimensional morphological assessment system to predict cancer drug-induced cardiotoxicity using human induced pluripotent stem cell-derived cardiomyocytes.

Recent developments of novel targeted therapies are contributing to the increased long-term survival of cancer patients; however, drug-induced cardiotoxicity induced by cancer drugs remains a serious problem in clinical settings. Nevertheless, there are few in vitro cell-based assays available to predict this toxicity, especially from the aspect of morphology. Here, we developed a simple two-dimensional (2D) morphological assessment system, 2DMA, to predict drug-induced cardiotoxicity in cancer patients using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with image-based high-content analysis in a high-throughput manner. To assess the effects of drugs on cardiomyocytes, we treated iPSC-CMs with 28 marketed pharmaceuticals and measured two key parameters: number of cell nuclei and sarcomere morphology. Drugs that significantly perturbed these two parameters at concentrations ≤30 times the human Cmax value were regarded as positive in the test. Based on these criteria, the sensitivity and specificity of the 2DMA system were 81% and 100%, respectively. Moreover, the translational predictability of 2DMA was comparable with that of a three-dimensional cardiotoxicity assay. RNA sequencing further revealed that the expression levels of several genes related to sarcomere components decreased following treatment with sunitinib, suggesting that inhibition of the synthesis of proteins that comprise the sarcomere contributes to drug-induced sarcomere disruption. Based on these features, the 2DMA system provides mechanistic insight with high predictability of cancer drug-induced cardiotoxicity in humans, and could thus contribute to the reduction of drug attrition rates at early stages of drug development.

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.

Brain activity changes in a macaque model of oxaliplatin-induced neuropathic cold hypersensitivity.

The antineoplastic agent oxaliplatin induces a painful peripheral neuropathy characterized by an acute cold hypersensitivity. There is a lack of effective treatments to manage oxaliplatin-induced cold hypersensitivity which is due, in part, to a lack of understanding of the pathophysiology of oxaliplatin-induced cold hypersensitivity. Thus, brain activity in oxaliplatin-treated macaques was examined using functional magnetic resonance imaging (fMRI). Oxaliplatin treatment reduced tail withdrawal latency to a cold (10 °C) stimulus, indicating cold hypersensitivity and increased activation in the secondary somatosensory cortex (SII) and the anterior insular cortex (Ins) was observed. By contrast, no activation was observed in these areas following cold stimulation in untreated macaques. Systemic treatment with an antinociceptive dose of the serotonergic-noradrenergic reuptake inhibitor duloxetine decreased SII and Ins activity. Pharmacological inactivation of SII and Ins activity by microinjection of the GABAA receptor agonist muscimol increased tail withdrawal latency. The current findings indicate that SII/Ins activity is a potential mediator of oxaliplatin-induced cold hypersensitivity.

Rationale for and Design of the PARADIGM Study: Randomized Phase III Study of mFOLFOX6 Plus Bevacizumab or Panitumumab in Chemotherapy-naïve Patients With RAS (KRAS/NRAS) Wild-type, Metastatic Colorectal Cancer.

BACKGROUND: It remains unclear whether an anti-VEGF or anti-EGFR antibody with standard doublet chemotherapy is the optimal first-line treatment in patients with RAS (KRAS/NRAS) wild-type metastatic colorectal cancer (mCRC). Here we outline the PARADIGM study (NCT02394795), designed to evaluate the superiority of panitumumab over bevacizumab, in combination with oxaliplatin/5-fluorouracil/leucovorin (mFOLFOX6) in patients with RAS wild-type chemotherapy-naïve mCRC. PATIENTS AND METHODS: Eligible patients are aged 20 to 79 years with an ECOG performance status of 0-1 and histologically/cytologically confirmed RAS wild-type mCRC. A total of 800 patients are to be randomly assigned (1:1 ratio) to mFOLFOX6 plus panitumumab (n = 400) or bevacizumab (n = 400) and stratified according to institution, age (20-64 vs. 65-79 years), and liver metastases (present vs. absent). Each treatment regimen includes oxaliplatin 85 mg/m2, l-leucovorin 200 mg/m2, and 5-fluorouracil (5-FU) I.V. 400 mg/m2 on day 1; 5-FU continuous I.V. 2400 mg/m2 on days 1 to 3; and either panitumumab 6 mg/kg or bevacizumab 5 mg/kg on day 1 every 2 weeks. The primary endpoint is overall survival forming the basis to detect a hazard ratio of 0.76 with a 1-sided type I error rate of 0.025 and 80% power. Secondary efficacy endpoints include progression-free survival, response rate, duration of response, and curative resection rate. A comprehensive biomarker analysis (NCT02394834) using archival tumor tissue and circulating tumor DNA samples collected at different time points (pretreatment and confirmed progressive disease) will investigate potential biomarkers related to primary and secondary resistance. The first patient was enrolled in May 2015 and the study is anticipated to complete in 2020.

Oxidation of DJ-1 induced by 6-hydroxydopamine decreasing intracellular glutathione.

DJ-1, the causative gene of a familial form of Parkinson's disease (PD), has been reported to undergo preferential oxidation of the cysteine residue at position 106 (Cys-106) under oxidative stress; however, details of the molecular mechanisms are not well known. In the present study, mechanisms of DJ-1 oxidation induced by 6-hydroxydopamine (6-OHDA) were investigated by using SH-SY5Y cells. The treatment of these cells with 6-OHDA caused an obvious acidic spot sift of DJ-1 due to its oxidation. However, when catalase, which is an hydrogen peroxide (H(2)O(2))-removing enzyme, was added during the treatment, it failed to prevent the oxidation induced by 6-OHDA, suggesting that electrophilic p-quinone formed from 6-OHDA, but not H(2)O(2), was responsible for the DJ-1 oxidation. Benzoquinone, another electrophilic p-quinone, also induced DJ-1 oxidation. The intracellular glutathione (GSH) levels were significantly decreased by 6-OHDA, irrespective of the presence or absence of catalase. The inhibition of GSH synthesis by buthionine sulfoximine resulted in a decrease in GSH levels and enhancement of DJ-1 oxidation. The pretreatment of cells with N-acetyl-cysteine prevented the loss of intracellular GSH and subsequently DJ-1 oxidation induced by 6-OHDA. Collectively, these results suggest that electrophilic p-quinone formed from 6-OHDA induces DJ-1 oxidation by decreasing intracellular GSH.

Coil embolization of a traumatic intracranial aneurysm of the distal part of the azygos anterior cerebral artery followed up for 4 years.

Traumatic intracranial aneurysms are rare, usually occur as a result of traffic accidents, and are associated with high rates of morbidity and mortality. A 12-year-old boy was admitted for treatment of cerebral hemorrhage in the left frontal lobe, 3 months after a traffic accident. Angiography revealed an intracranial aneurysm of the distal part of the azygos anterior cerebral artery (ACA). The maximum size of this aneurysm was 9.5 mm. Coil embolization was successfully performed with preservation of the parent arteries. Angiography performed at the 4-year follow-up examination revealed no abnormalities. The present case showed that primary coiling may allow aneurysm occlusion along with parent vessel preservation in selected cases of traumatic intracranial aneurysm of the distal part of the azygos ACA.

24(S)-hydroxycholesterol induces neuronal cell death through necroptosis, a form of programmed necrosis.

24(S)-Hydroxycholesterol (24S-OHC) produced by cholesterol 24-hydroxylase expressed mainly in neurons plays an important physiological role in the brain. Conversely, it has been reported that 24S-OHC possesses potent cytotoxicity. The molecular mechanisms of 24S-OHC-induced cell death have not yet been fully elucidated. In this study, using human neuroblastoma SH-SY5Y cells and primary cortical neuronal cells derived from rat embryo, we characterized the form of cell death induced by 24S-OHC. SH-SY5Y cells treated with 24S-OHC exhibited neither fragmentation of the nucleus nor caspase activation, which are the typical characteristics of apoptosis. 24S-OHC-treated cells showed necrosis-like morphological changes but did not induce ATP depletion, one of the features of necrosis. When cells were treated with necrostatin-1, an inhibitor of receptor-interacting serine/threonine kinase 1 (RIPK1) required for necroptosis, 24S-OHC-induced cell death was significantly suppressed. The knockdown of RIPK1 by transfection of small interfering RNA of RIPK1 effectively attenuated 24S-OHC-induced cell death. It was found that neither SH-SY5Y cells nor primary cortical neuronal cells expressed caspase-8, which was regulated for RIPK1-dependent apoptosis. Collectively, these results suggest that 24S-OHC induces neuronal cell death by necroptosis, a form of programmed necrosis.

Brain abscess following intra-arterial thrombolytic treatment for acute brain ischemia.

Brain abscess formation is a rare complication of intracranial endovascular treatment. To our knowledge, all previous reports of brain abscess formation have been associated with treatments involving the introduction of foreign materials. A 75-year-old man was admitted to hospital for acute stroke. Cerebral angiography revealed occlusion of the left middle cerebral artery (MCA) at the origin of the M2 segment. Intra-arterial thrombolytic therapy was administered but a hemorrhagic event occurred during this process. A brain CT scan revealed a hematoma extending from the left basal ganglia to the left frontal lobe and expansion of the infarct in the left MCA territory. A brain abscess at the hemorrhagic site developed 3 months after symptom onset. This is the first report of a patient with brain abscess formation following intra-arterial thrombolytic treatment. It is important to ensure aseptic technique during endovascular procedures irrespective of the involvement of foreign materials.

Cytoprotective effects of vitamin E homologues against glutamate-induced cell death in immature primary cortical neuron cultures: Tocopherols and tocotrienols exert similar effects by antioxidant function.

Glutamate plays a critical role in pathological cell death within the nervous system. Vitamin E is known to protect cells from glutamate cytotoxicity, either by direct antioxidant action or by indirect nonantioxidant action. Further, α-tocotrienol (α-T3) has been reported to be more effective against glutamate-induced cytotoxicity than α-tocopherol (α-T). To shed more light on the function of vitamin E against glutamate toxicity, the protective effects of eight vitamin E homologues and related compounds, 2,2,5,7,8-pentamethyl-6-chromanol (PMC) and 2-carboxy-2,5,7,8-pentamethyl-6-chromanol (Trolox), against glutamate-induced cytotoxicity on immature primary cortical neurons were examined using different protocols. Glutamate induced the depletion of glutathione and generation of reactive oxygen species and lipid hydroperoxides, leading to cell death. α-, ß-, γ-, and δ-T and -T3; PMC; and Trolox all exerted cytoprotective effects against glutamate-induced cytotoxicity, and a longer preincubation time increased both the cellular content and the cytoprotective effects of T more significantly than those of T3, the effect of preincubation being relatively small for T3 and PMC. The protective effect of Trolox was less potent than that of PMC. The cytoprotective effects of α-T and α-T3 corresponded to their intracellular content. Further, lipid peroxidation products were measured after reduction with triphenylphosphine followed by saponification with potassium hydroxide. It was found that glutamate treatment increased the formation of hydroxyeicosatetraenoic acid, hydroxyoctadecadienoic acid, and 8-F(2)-isoprostane 2α, which was suppressed by α-T. This study shows that vitamin E protects cells from glutamate-induced toxicity primarily by direct antioxidant action and that the apparent higher capacity of T3 compared to T is ascribed to the faster uptake of T3 compared to T into the cells. It is suggested that, considering the bioavailability, α-T should be more effective than α-T3 against glutamate toxicity in vivo.

Small-molecule inhibition of Wee1 kinase by MK-1775 selectively sensitizes p53-deficient tumor cells to DNA-damaging agents.

Wee1 is a tyrosine kinase that phosphorylates and inactivates CDC2 and is involved in G(2) checkpoint signaling. Because p53 is a key regulator in the G(1) checkpoint, p53-deficient tumors rely only on the G(2) checkpoint after DNA damage. Hence, such tumors are selectively sensitized to DNA-damaging agents by Wee1 inhibition. Here, we report the discovery of a potent and selective small-molecule inhibitor of Wee1 kinase, MK-1775. This compound inhibits phosphorylation of CDC2 at Tyr15 (CDC2Y15), a direct substrate of Wee1 kinase in cells. MK-1775 abrogates G(2) DNA damage checkpoint, leading to apoptosis in combination with DNA-damaging chemotherapeutic agents such as gemcitabine, carboplatin, and cisplatin selectively in p53-deficient cells. In vivo, MK-1775 potentiates tumor growth inhibition by these agents, and cotreatment does not significantly increase toxicity. The enhancement of antitumor effect by MK-1775 was well correlated with inhibition of CDC2Y15 phosphorylation in tumor tissue and skin hair follicles. Our data indicate that Wee1 inhibition provides a new approach for treatment of multiple human malignancies.

Discovery of gene expression-based pharmacodynamic biomarker for a p53 context-specific anti-tumor drug Wee1 inhibitor.

BACKGROUND: Wee1 is a tyrosine kinase regulating S-G2 cell cycle transition through the inactivating phosphorylation of CDC2. The inhibition of Wee1 kinase by a selective small molecule inhibitor significantly enhances the anti-tumor efficacy of DNA damaging agents, specifically in p53 negative tumors by abrogating S-G2 checkpoints, while normal cells with wild-type p53 are not severely damaged due to the intact function of the G1 checkpoint mediated by p53. Since the measurement of mRNA expression requires a very small amount of biopsy tissue and is highly quantitative, the development of a pharmacodynamic (PD) biomarker leveraging mRNA expression is eagerly anticipated in order to estimate target engagement of anti-cancer agents. RESULTS: In order to find the Wee1 inhibition signature, mRNA expression profiling was first performed in both p53 positive and negative cancer cell lines treated with gemcitabine and a Wee1 inhibitor, MK-1775. We next carried out mRNA expression profiling of skin samples derived from xenograft models treated with the Wee1 inhibitor to identify a Wee1 inhibitor-regulatory gene set. Then, the genes that were commonly modulated in both cancer cell lines and rat skin samples were extracted as a Wee1 inhibition signature that could potentially be used as a PD biomarker independent of p53 status. The expression of the Wee1 inhibition signature was found to be regulated in a dose-dependent manner by the Wee1 inhibitor, and was significantly correlated with the inhibition level of a direct substrate, phosphorylated-CDC2. Individual genes in this Wee1 inhibition signature are known to regulate S-G2 cell cycle progression or checkpoints, which is consistent with the mode-of-action of the Wee1 inhibitor. CONCLUSION: We report here the identification of an mRNA gene signature that was specifically changed by gemcitabine and Wee1 inhibitor combination treatment by molecular profiling. Given the common regulation of expression in both xenograft tumors and animal skin samples, the data suggest that the Wee1 inhibition gene signature might be utilized as a quantitative PD biomarker in both tumors and surrogate tissues, such as skin and hair follicles, in human clinical trials.

Expression levels of NF-Y target genes changed by CDKN1B correlate with clinical prognosis in multiple cancers.

CDK inhibitors CDKN1B (p27) and CDKN2A (p16) inhibit cell cycle progression. A lower expression level of only p27 has been correlated with poorer prognosis in various types of clinical cancers. The difference may be the result of distinct genes downstream of these CDK inhibitors. Here, we report that NF-Y transcription factor-targeted genes specifically down-regulated by p27 correlate with poor prognosis in multiple tumor types. We performed mRNA expression profiling in HCT116 cells over-expressing either p16 or p27 and identified their regulatory genes. In silico transcription factor prediction indicated that most of the genes specifically down-regulated by p27 are controlled by NF-Y. Under the hypothesis that NF-Y-targeted genes are responsible for poor prognosis, we predicted prognosis in four types of cancer based on genes with the NF-Y motif, and found a significant association between the expression of NF-Y-targeted genes and poor prognosis.