Phosphoprotein Levels Research Articles

Development and characterisation of a panel of phosphatidylinositide 3-kinase \u2013 mammalian target of rapamycin inhibitor resistant lung cancer cell lines

The PI3K-mTOR pathway is involved in regulating all hallmarks of cancer, and is often dysregulated in NSCLC, making it an attractive therapeutic target in this setting. Acquired resistance to PI3K-mTOR inhibition is a major hurdle to overcome in the success of PI3K-mTOR targeted agents. H460, A549, and H1975 resistant cells were generated by prolonged treatment in culture with Apitolisib (GDC-0980), a dual PI3K-mTOR inhibitor over a period of several months, from age-matched parent cells. Resistance was deemed to have developed when a log fold difference in IC50 had been achieved. Resistant cell lines also exhibited resistance to another widely investigated PI3K-mTOR dual inhibitor; Dactolisib (BEZ235). Cell lines were characterised at the level of mRNA (expression array profiling expression of >150 genes), miRNA (expression array profiling of 2100 miRNAs), protein (bottoms-up label-free mass spectrometry) and phosphoprotein (expression array profiling of 84 phospho/total proteins). Key alterations were validated by qPCR and Western blot. H1975 cells were initially most sensitive to Apitolisib (GDC-0980), but developed resistance more quickly than the other cell lines, perhaps due to increased selective pressure from the impressive initial effect. In-depth molecular profiling suggested epithelial-mesenchymal transition (EMT) may play a role in resistance to PI3K-mTOR dual inhibition in NSCLC.

Positive associations between upregulated levels of stress-induced phosphoprotein 1 and matrix metalloproteinase-9 in endometriosis/adenomyosis.

Stress-induced phosphoprotein-1 (STIP1), an adaptor protein that coordinates the functions of HSP70 and HSP90 in protein folding, has been implicated in the development of human gynecologic malignancies. This case-control study investigates STIP1 serum levels and tissue expression in relation to endometriosis/adenomyosis in Taiwanese population. Female patients with surgically confirmed endometriosis/adenomyosis were compared with women free of endometriosis/adenomyosis. Serum STIP1 levels were measured using an enzyme-linked immunosorbent assay and surgical tissues were analyzed by immunohistochemistry. Both epithelial and stromal cells in surgical tissues of endometriosis and adenomyosis expressed STIP1 and MMP-9. Notably, MMP-9 expression was significantly decreased when STIP1 expression was knocked-down. In vitro experiments revealed that STIP1 was capable of binding to the MMP-9 promoter and enhanced its transcriptional expression. The preoperative serum STIP1 levels of patients with endometriosis/adenomyosis were significantly higher than those of the controls. In brief, our data suggest an association between STIP1 levels and endometriosis/adenomyosis.

Single cell profiling of phospho-protein levels in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) has a high incidence and a steeply growing prevalence in the Western world. The heterogeneity of the disease necessitates individual mapping of biology and predicted drug response in each patient as basis for administration of tailored treatments. Cell signaling aberrations may serve as biological indicators for suitable therapy. By applying phospho-specific flow cytometry, we mapped basal and induced phosphorylation levels of 20 phospho-epitopes on proteins relevant to B-cell signaling in B cells from 22 CLL patients and 25 normal controls. The signaling response of the cytostatic drugs fludarabine, doxorubicin and vincristine was also investigated. CLL cells exerted similar or lower basal phosphorylation levels compared to normal B cells, with the exception of STAT3 (pY705) which was increased. Interestingly, STAT3 inhibitors normalized the STAT3 (pY705) level and reduced cell viability. Vincristine treatment significantly modulated phosphorylation levels in CLL cells, while no effect was observed in controls or after fludarabine or doxorubicin treatment. After BCR stimulation, CLL cells showed a tendency towards impaired phosphorylation levels, significant for several of the analyzed proteins. However, the level of Akt (pS473) was more potently induced in IgHV unmutated CLL (UM-CLL) patient samples and was significantly higher than in M-CLL samples. Importantly, the PI3Kδ inhibitor idelalisib potently reversed the effect of anti-IgM on Akt (pS473). Thus, signaling aberrations could be identified by phosphoflow cytometry and aberrant signaling could be normalized by small molecule drugs. This approach can identify relevant drug targets as well as drug effects in the individual patient.

Bubbles Induce Endothelial Microparticle Formation via a Calcium-Dependent Pathway Involving Flippase Inactivation and Rho Kinase Activation

Background/Aims: Intravascular bubbles can exert pleiotropic detrimental effects, partly by inducing endothelial microparticles (EMPs) production, which play critical roles in cell communication and vascular inflammation cascades. However, the underlying mechanisms remain unclear. This study aimed to delineate the possible mechanisms involving bubble-induced EMPs formation. Methods: Human umbilical vein endothelial cells (HUVECs) were contacted by bubbles and EMPs level in supernatant were quantified by flow cytometry. Cytoplasmic calcium (Ca²⁺) was measured by the Ca²⁺ binding dyes Fluo-3 AM and flippase activity was assessed by translocation rate of fluorescent phosphatidylserine (PS) analogue NBD-PS. Protein levels of phospho-myosin light chain (MLC, a Rho kinase substrate) and phospho-extracellular signal-regulated kinase 1 or 2 (ERK1/2) were determined by western blotting. The score of actin colocalization was assessed by phalloidin-FITC using an immunofluorescent microscopy. Results: EMPs level markedly increased after bubble stimulus. Cytoplasmic calcium (Ca²⁺) significantly elevated (P< 0.05), flippase activity decreased (P< 0.05), protein levels of phospho- MLC and phospho- ERK1/2 significantly increased (P< 0.05, P < 0.05), and the score of actin colocalization markedly reduced (P< 0.05) in bubble-stimulated HUVECs. All the above changes except the increase in phospho-ERK1/2 can be reversed by Ca²⁺ channel blocker LaCl₃ (P< 0.05). Additionally, MLC phosphorylation was significantly inhibited and actin colocalization markedly increased by Rho kinase inhibitor pretreatment and more importantly, bubble-induced EMPs markedly decreased. Conclusions: These results demonstrate that bubble stimulates EMPs formation by cytoplasmic Ca²⁺ elevation and subsequently activating Rho kinase pathway and cytoskeleton reorganization. Simultaneously, cytoplasmic Ca²⁺ inhibits the flippase activity and subsequently increases phosphatidylserine exposure, which also contributes to EMPs formation.

Sample Preparation for Relative Quantitation of Proteins Using Tandem Mass Tags (TMT) and Mass Spectrometry (MS).

Quantitative proteome analysis allows comparisons of protein or phosphoprotein levels across multiple cell types or conditions. A number of experimental approaches have been described toward quantitative proteomics. In this chapter, we focus on Tandem Mass Tag (TMT) isobaric labeling of peptides for global, relative quantitation of proteins and phosphopeptides. To date, there has been no published protocol describing chemical labeling of small amounts of peptides specifically extracted from small tumor samples, for which rigorous sample preparation is necessary to ensure reproducible TMT labeling.

Correlation of CT indicators of NSCLC and pathological features and the expression level of p53 and c-myc.

To investigate the correlations of the computed tomography (CT) signs of non-small cell lung cancer (NSCLC) with pathological features and the expression levels of phosphoprotein 53 (p53) and c-Myc in patients. 87 patients with NSCLC admitted to the Department of Oncology in our hospital from July 2014 to March 2017 were randomly selected. Morphologies of NSCLC and cancer-adjacent tissues were detected by hematoxylin and eosin (H&E) staining; messenger ribonucleic acid (mRNA) and protein levels of p53 and c-Myc in cancer and cancer-adjacent tissues were detected using real-time polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC); spiral CT (SCT) was conducted for exploring imaging signs of patients with NSCLC; the correlation of CT signs with pathology and the expressions of p53 and c-Myc was analyzed. H&E staining showed that NSCLC tissues had a larger nucleus, a larger nucleus-cytoplasm ratio, and a more evident histopathological atypia, with no clear histological structure compared with cancer-adjacent normal tissues; RT-PCR and IHC results revealed that the mRNA and protein expression levels of p53 and c-Myc in NSCLC tissues were significantly higher than those in cancer-adjacent tissues, in which differences in mRNA levels were 1.75 folds and 1.84 folds, respectively (p<0.05). SCT signs indicated that swollen lymph nodes and spiculation, spinous process and deep lobulation signs often occurred in the chest of NSCLC patients, and pleural indentation appeared in the majority of patients; the chi-square test results showed that the positive rates of p53 and c-Myc proteins were not related to pathological types of NSCLC, but significantly correlated with tumor differentiation (p<0.05); the positive rates of p53 and c-Myc proteins were correlated with tumor diameter, spiculation and deep lobulation signs and lymph node metastasis (p<0.05), but not associated with spinous process, vacuole and pleural indentation signs (p>0.05). CT scan combined with the detection of p53 and c-Myc expressions can improve the diagnosis of lymph node metastasis and clinical staging for patients with NSCLC, which is conducive to the clinical treatment and prognosis analysis of NSCLC.

Proteomics and phosphoproteomics analysis of liver in male rats exposed to bisphenol A: Mechanism of hepatotoxicity and biomarker discovery

Bisphenol A (BPA), discovered to be an artificial estrogen, has been shown to leach from some containers and mediate oxidative damage to cells and tissues and to be involved in reproductive disorders, obesity, diabetes, and liver dysfunction. In the current study, we investigated the effects of oral chronic exposure to low dose of BPA (0.5 mg kg−1) on the protein and phosphoprotein expression profiles in male Wistar rat liver using a gel-based proteomics approach based on two-dimensional gel electrophoresis followed by matrix-assisted laser desorption/ionization mass spectrometry identification. Our results showed that BPA exposure affected the levels of proteins and phosphoproteins involved in diverse biological processes associated with hepatotoxicity, fatty liver, and carcinoma. Moreover, we analyzed the effects of BPA on oxidative stress by assessing levels of malondialdehyde (MDA), a marker of lipid peroxidation, and reduced glutathione (GSH), a non-enzymatic antioxidant agent, in the liver. As expected BPA induced oxidative stress indicated by increased levels of MDA and decreased GSH content in the liver. In conclusion, chronic oral exposure of rats to BPA leads to increased oxidative stress in the liver and major alterations in the liver proteome and phosphoproteome, which may contribute to the pathophysiology of liver diseases.

Cannabidiol and palmitoylethanolamide are anti-inflammatory in the acutely inflamed human colon.

We sought to quantify the anti-inflammatory effects of two cannabinoid drugs, cannabidiol (CBD) and palmitoylethanolamide (PEA), in cultured cell lines and compared this effect with experimentally inflamed explant human colonic tissue. These effects were explored in acutely and chronically inflamed colon, using inflammatory bowel disease and appendicitis explants. Caco-2 cells and human colonic explants collected from elective bowel cancer, inflammatory bowel disease (IBD) or acute appendicitis resections, and were treated with the following drug treatments: vehicle, an inflammatory protocol of interferon γ (IFNγ) and tumour necrosis factor α (TNFα; 10 ng/ml), inflammation and PEA (10 µM), inflammation and CBD (10 µM), and PEA or CBD alone, CBD or vehicle were added simultaneously with IFNγ. Nine intracellular signalling phosphoproteins were determined by multiplex. Inflammatory cytokine secretion was determined using ELISA. Receptor mechanisms were investigated using antagonists for CB1, CB2, PPARα, PPARγ, TRPV1 and GPR55. IFNγ and TNFα treatment increased phosphoprotein and cytokine levels in Caco-2 cultures and colonic explants. Phosphoprotein levels were significantly reduced by PEA or CBD in Caco-2 cultures and colonic explants. CBD and PEA prevented increases in cytokine production in explant colon, but not in Caco-2 cells. CBD effects were blocked by the CB2 antagonist AM630 and TRPV1 antagonist SB366791. PEA effects were blocked by the PPARα antagonist GW6471. PEA and CBD were anti-inflammatory in IBD and appendicitis explants. PEA and CBD are anti-inflammatory in the human colon. This effect is not seen in cultured epithelial cells. Appropriately sized clinical trials should assess their efficacy.

Correction: Decoupling of the PI3K Pathway via Mutation Necessitates Combinatorial Treatment in HER2+ Breast Cancer.

[This corrects the article DOI: 10.1371/journal.pone.0133219.].

Abnormalities of signal transduction networks in chronic schizophrenia

Schizophrenia is a serious neuropsychiatric disorder characterized by disruptions of brain cell metabolism, microstructure, and neurotransmission. All of these processes require coordination of multiple kinase-mediated signaling events. We hypothesize that imbalances in kinase activity propagate through an interconnected network of intracellular signaling with potential to simultaneously contribute to many or all of the observed deficits in schizophrenia. We established a workflow distinguishing schizophrenia-altered kinases in anterior cingulate cortex using a previously published kinome array data set. We compared schizophrenia-altered kinases to haloperidol-altered kinases, and identified systems, functions, and regulators predicted using pathway analyses. We used kinase inhibitors with the kinome array to test hypotheses about imbalance in signaling and conducted preliminary studies of kinase proteins, phosphoproteins, and activity for kinases of interest. We investigated schizophrenia-associated single nucleotide polymorphisms in one of these kinases, AKT, for genotype-dependent changes in AKT protein or activity. Kinome analyses identified new kinases as well as some previously implicated in schizophrenia. These results were not explained by chronic antipsychotic treatment. Kinases identified in our analyses aligned with cytoskeletal arrangement and molecular trafficking. Of the kinases we investigated further, AKT and (unexpectedly) JNK, showed the most dysregulation in the anterior cingulate cortex of schizophrenia subjects. Changes in kinase activity did not correspond to protein or phosphoprotein levels. We also show that AKT single nucleotide polymorphism rs1130214, previously associated with schizophrenia, influenced enzyme activity but not protein or phosphoprotein levels. Our data indicate subtle changes in kinase activity and regulation across an interlinked kinase network, suggesting signaling imbalances underlie the core symptoms of schizophrenia.

Ethanol alters N-methyl-D-aspartate receptor regulation in the hippocampus of adolescent rats.

Our laboratory and others have shown that ethanol disrupts hippocampus-associated memory formation in adolescent rats, but the signaling pathway involved remains largely unknown. Phosphorylation of the N-methyl-D-aspartate receptor, particularly the N-methyl-D-aspartate receptor subunit 2B (NR2B), has been implicated in several ethanol effects and in memory formation. The present study investigated the effects of repeated ethanol exposure on the phosphorylation of the NR2B subunit and src protein in the hippocampus of adolescent rats. Rats were treated daily with ethanol or vehicle for 5 consecutive days and sacrificed at several time points after the last treatment. At 30 min postethanol treatment, levels of phosphorylated NR2B and src were significantly elevated; this was the same postethanol time point when adolescent rats showed impairments in their performance of memory tasks. Peak increases in both phosphorylated levels of NR2B and src were seen around 1 h post-treatment, after which levels of both phosphoproteins started to decline. Together, these data suggest that the time-dependent increase in hippocampal phosphorylated NR2B and src levels following repeated ethanol exposure may be responsible for hippocampus-associated memory impairments in adolescent rats.

BDNF over-expression induces striatal serotonin fiber sprouting and increases the susceptibility to l-DOPA-induced dyskinesia in 6-OHDA-lesioned rats

In addition to its role in neuronal survival, the brain neurotrophic factor (BDNF) has been shown to influence serotonin transmission and synaptic plasticity, events strongly implicated in the appearance of l-DOPA-induced dyskinesia (LID), a motor complication occurring in parkinsonian patients after long-term treatment with the dopamine precursor.In order to evaluate a possible influence of BDNF in the appearance of LID, 6-OHDA-lesioned rats received a striatal injection of different concentrations of an adeno-associated viral (AAV) vector over-expressing either BDNF or GFP, as control vector. Eight weeks later, animals started to receive a daily treatment with l-DOPA (4–6mg/kg plus benserazide 4–6mg/kg, s.c.) or saline, and dyskinesias, as well as l-DOPA-induced rotations, were evaluated at several time-points. Moreover, molecular changes in striatal D1 receptor-dependent cAMP/PKA and ERK/mTORC signaling pathways, as well as, sprouting of striatal serotonin axons, were measured. Results showed that the AAV-BDNF vector injection induced striatal over-expression of BDNF, as well as striatal and pallidal serotonin axon hyperinnervation. Moreover, rats that over-expressed BDNF were more prone to develop LID and l-DOPA-induced rotations, compared to the GFP-treated control group. Finally, rats that over-expressed BDNF showed increased levels of striatal D1R-dependent signaling phospho-proteins in response to l-DOPA administration. This study suggests that BDNF over-expression, by inducing changes in pre-synaptic serotonin axonal trophism, is able to exacerbate maladaptive responses to l-DOPA administration.

Potential anti-cancer activity of 7-O-pentyl quercetin: Efficient, membrane-targeted kinase inhibition and pro-oxidant effect

Quercetin is a redox-active plant-derived flavonoid with potential anticancer effects, stemming largely from its interaction with a number of proteins, and in particular from inhibition of pro-life kinases. To improve efficacy, we reasoned that a local increase in concentration of the compound at the level of cell membranes would result in a more efficient interaction with membrane-associated signaling kinases. We report here the synthesis of all five isomeric quercetin derivatives in which an n-pentyl group was linked via an ether bond to each hydroxyl of the flavonoid kernel. This strategy proved effective in directing quercetin to cellular membranes, and revealed a remarkable dependence of the derivatives’ bioactivity on the specific site of functionalization. The isomer bearing the pentyl group in position 7, Q-7P, turned out to be the most effective and promising derivative, selectively inducing apoptosis in tumoral and fast-growing cells, while sparing slow-growing, non-tumoral ones. Cytotoxicity for tumoral cells was strongly enhanced compared to quercetin itself. Q-7P induced massive ROS production, which however accounted only partially for cell death. Alterations in the levels of various signaling phospho-proteins were observed in a proteomics screen. An important contribution seems to come from inhibition of the PI3K/Akt pathway. This work opens new perspectives in developing membrane-associating, polyphenol-based anticancer agents.

Abstract 3717: New therapies for the treatment of BRAF/NRAS wild type melanoma

Abstract Melanoma represents the common tumor whose incidence has increased the most in the last 30 years and causes more than one death every hour in the US alone. Despite significant advances in targeted and immunotherapies, most patients cannot still be cured. Our aim is to identify new drug combinations that are synergistic in BRAF/NRAS wild type melanoma, a sub-type representing 30% of cases for which targeted therapies are not currently available. We high-throughput screened a collection of 20 BRAF/NRAS wild type melanoma cell lines with 180 drug combinations (60 library drugs used at 5 different concentrations combined with 3 clinically relevant anchor drugs) and generated over 8000 survival curves . We found that 25% of cell lines are highly sensitive to a combination of nilotinib plus trametinib and confirmed this finding with 2 independent assays. We further validated the drug synergy firstly using an independent collection of BRAF/NRAS wild type melanoma cell lines (n=7), then a collection of BRAF/NRAS wild type patient derived xenotransplant cultures (n=3), and finally with a collection of BRAFV600E and NRASQ61 melanoma cell lines (n=12). Further, we generated a gene expression signature of cell lines that display synergy for the nilotinib/trametinib combination, and used it to classify human melanomas from Leeds Melanoma Project (N=171) and TCGA (n=470) cohorts. Tumors classified as “synergistic-like” (27.9 and 36.7%, respectively) are associated to decreased overall and recurrence free survival (P&amp;lt;0.05), suggesting that our combination might be effective in a relevant fraction of aggressive tumors. In order to identify drug resistance mechanisms we deployed a genome-wide CRISPR/Cas9 screen. We found that loss of the tuberous sclerosis complex can confer resistance to nilotinib/trametinib, and validated this mechanism using clonal engineered lines. Since tuberous sclerosis complex genes are mutated in 10% of melanomas, this approach can help to identify patients potentially refractory to the treatment. We also investigated the molecular mechanism of nilotinib/trametinib synergy by analysing the level of several phosphoproteins upon treatment. We discovered that the nilotinib/trametinib combination synergistically reduce the level of P-ERK in synergistic cell lines but not in cell lines resistant to the drug combination, thus pointing out the MAPK pathway dependence of the synergy. This finding provides a putative marker to identify tumors responsive to the treatment. Finally, we tested in vivo the nilotinib/trametinib combination in a patient derived xenotransplant mouse model and showed that the combination is well tolerated and significantly more effective than the 2 drugs alone (P&amp;lt;0.01). These data suggest a strong clinical translation potential for nilotinib/trametinib combination and pave the way to the development of clinical trials for BRAF/NRAS wild type melanoma. Citation Format: Marco Ranzani, Kristel Kemper, Magali Michaut, Oscar Krijgsman, Vivek Iyer, Anneliese Speak, Jeremie Nsengimana, Kim Wong, Vera Grinkevich, Nanne Aben, Martin Del Castillo Velasco-Herrera, Clara Alsinet, Marcela Sjoberg, Mamunur Rashid, Gemma Turner, Fiona Behan, Emmanuelle Supper, Nicola Thompson, Graham Bignell, Ken Dutton-Regester, Antonia Pritchard, Chi Wong, Ultan McDermott, Nicholas K. Hayward, Kosuke Yusa, Julia Newton-Bishop, Lodewyk Wessels, Mathew Garnett, Daniel Peeper, David Adams. New therapies for the treatment of BRAF/NRAS wild type melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3717. doi:10.1158/1538-7445.AM2017-3717

Increased PDGFR-beta and VEGFR-2 protein levels are associated with resistance to platinum-based chemotherapy and adverse outcome of ovarian cancer patients.

Despite frequent initial response rates of epithelial ovarian cancer to platinum-based chemotherapy the majority of patients develop drug resistance. Our aim was to evaluate differential expression of signaling-pathway proteins in platinum-sensitive versus platinum-resistant primary epithelial ovarian cancer specimens to identify predictive biomarkers for treatment response.192 patients were studied comprising of independent training (n = 89) and validation (n = 103) cohorts. Full-length proteins were extracted from paraffin-embedded samples including multiple regions per tumor to account for intratumoral heterogeneity. Quantitative reverse-phase-protein-arrays were used to analyze protein and phospho-protein levels of 41 signaling molecules including growth-factor receptors, AKT and MAPK signaling pathways as well as angiogenesis and cell-adhesion.Platinum-resistant ovarian cancers (56/192) demonstrated significantly higher intratumoral levels of the angiogenesis-associated growth-factor receptors PDGFR-beta and VEGFR2 compared to platinum-sensitive tumors. In addition, patients with high PDGFR-beta expression had significantly shorter overall and progression-free survival (HR 3.6 and 2.4; p < 0.001). The prognostic value of PDGFR-beta and VEGFR2 was confirmed in publicly available microarray-datasets.High intratumoral levels of the angiogenesis-related growth-factor receptors PDGFR-beta and VEGFR2 might serve as novel predictive biomarkers to identify primary resistance to platinum-based chemotherapy. Those ovarian cancer patients might particularly benefit from additional anti-vascular therapy including anti-VEGF antibody or receptor tyrosine-kinase-inhibitor therapy.

Butein inhibits NF-κB, AP-1 and Akt activation in adult T-cell leukemia/lymphoma.

Human T-cell leukemia virus type 1(HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma(ATLL) but there is no effective treatment for HTLV-1-associated diseases. Herein, we determined the effect of butein, a bioactive plant polyphenol, on cell growth, apoptosis and signaling pathways in HTLV-1-infected T-cell lines and on tumor growth in SCID mice. Treatment with butein caused a decrease in viability of HTLV-1-infected T-cell lines. Tcells cultured with butein showed obvious apoptosis morphology, and cleavage of poly(ADP-ribose) polymerase with activation of caspase-3, -8 and -9. Pretreatment of cells with caspase inhibitor partially blocked butein-induced inhibition of cell viability. Butein also resulted in cell cycle arrest at G1phase. Butein markedly downregulated the protein expression levels of CDK4, CDK6, cyclin D1, cyclin D2, cyclin E, survivin, XIAP, c-IAP2 and phospho-pRb. Butein also inhibited i) total and phospho-protein levels of IκB kinase (IKK)α and IKKβ, ii) degradation and phosphorylation of IκBα, iii) JunB and JunD, iv) total and phospho-protein levels of Akt, v) phosphorylation of RelA, vi)heat shock protein90, and vii) DNA-binding activity of NF-κB and AP-1. In mice harboring ATLL xenograft tumors, butein caused a significant inhibition of tumor growth and reduced serum levels of soluble interleukin-2 receptorα chain and soluble cluster of differentiation30. Considered together, the results indicated that butein has antiproliferative and proapoptotic properties through the suppression of NF-κB, AP-1 and Akt signaling in HTLV-1-infected Tcells, both invitro and invivo, suggesting its therapeutic potential against HTLV-1-associated diseases including ATLL.

YAP/TAZ-Mediated Upregulation of GAB2 Leads to Increased Sensitivity to Growth Factor-Induced Activation of the PI3K Pathway.

The transcription regulators YAP and TAZ function as effectors of the HIPPO signaling cascade, critical for organismal development, cell growth, and cellular reprogramming, and YAP/TAZ is commonly misregulated in human cancers. The precise mechanism by which aberrant YAP/TAZ promotes tumor growth remains unclear. The HIPPO tumor suppressor pathway phosphorylates YAP and TAZ, resulting in cytosolic sequestration with subsequent degradation. Here, we report that the PI3K/AKT pathway, which is critically involved in the pathophysiology of endometrial cancer, interacts with the HIPPO pathway at multiple levels. Strikingly, coordinate knockdown of YAP and TAZ, mimicking activation of the HIPPO pathway, markedly decreased both constitutive and growth factor-induced PI3K pathway activation by decreasing levels of the GAB2 linker molecule in endometrial cancer lines. Furthermore, targeting YAP/TAZ decreased endometrial cancer tumor growth in vivo In addition, YAP and TAZ total and phosphoprotein levels correlated with clinical characteristics and outcomes in endometrial cancer. Thus, YAP and TAZ, which are inhibited by the HIPPO tumor suppressor pathway, modify PI3K/AKT pathway signaling in endometrial cancer. The cross-talk between these key pathways identifies potential new biomarkers and therapeutic targets in endometrial cancer. Cancer Res; 77(7); 1637-48. ©2017 AACR.

Interleukin-17 augments tumor necrosis factor α-mediated increase of hypoxia-inducible factor-1α and inhibits vasodilator-stimulated phosphoprotein expression to reduce the adhesion of breast cancer cells.

Interleukin-17 (IL-17) and tumor necrosis factor (TNF)-α are able to cooperatively alter the expression levels of a number of genes. In the present study, the mRNA expression levels of hypoxia-inducible factor (HIF)-1α were analyzed in MDA-MB-231 breast cancer cells following treatment with IL-17, TNF-α or the combination of IL-17 and TNF-α. The protein expression levels of HIF-1α and vasodilator-stimulated phosphoprotein (VASP) were evaluated using western blot analysis. The adhesive ability of the cells was determined using an MTT assay following treatment with HIF-1α-small interfering RNA and short hairpin RNA-VASP that were used to suppress the expression levels of HIF-1α and VASP protein, respectively. These results demonstrated that IL-17 augmented TNF-α-induced gene expression of HIF-1α. The combination of IL-17 and TNF-α promoted an increase in HIF-1α expression and a decrease in VASP expression and a reduction in the adhesive ability of cells. These results demonstrated that IL-17 effectively enhanced the TNF-α-induced increase in HIF-1α and inhibited VASP expression, thus reducing the adhesion of MDA-MB-231 cells.

Adaptive Multiview Nonnegative Matrix Factorization Algorithm for Integration of Multimodal Biomedical Data.

The amounts and types of available multimodal tumor data are rapidly increasing, and their integration is critical for fully understanding the underlying cancer biology and personalizing treatment. However, the development of methods for effectively integrating multimodal data in a principled manner is lagging behind our ability to generate the data. In this article, we introduce an extension to a multiview nonnegative matrix factorization algorithm (NNMF) for dimensionality reduction and integration of heterogeneous data types and compare the predictive modeling performance of the method on unimodal and multimodal data. We also present a comparative evaluation of our novel multiview approach and current data integration methods. Our work provides an efficient method to extend an existing dimensionality reduction method. We report rigorous evaluation of the method on large-scale quantitative protein and phosphoprotein tumor data from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) acquired using state-of-the-art liquid chromatography mass spectrometry. Exome sequencing and RNA-Seq data were also available from The Cancer Genome Atlas for the same tumors. For unimodal data, in case of breast cancer, transcript levels were most predictive of estrogen and progesterone receptor status and copy number variation of human epidermal growth factor receptor 2 status. For ovarian and colon cancers, phosphoprotein and protein levels were most predictive of tumor grade and stage and residual tumor, respectively. When multiview NNMF was applied to multimodal data to predict outcomes, the improvement in performance is not overall statistically significant beyond unimodal data, suggesting that proteomics data may contain more predictive information regarding tumor phenotypes than transcript levels, probably due to the fact that proteins are the functional gene products and therefore a more direct measurement of the functional state of the tumor. Here, we have applied our proposed approach to multimodal molecular data for tumors, but it is generally applicable to dimensionality reduction and joint analysis of any type of multimodal data.

Transient receptor potential melastatin (TRPM) 8 is expressed in freshly isolated native human odontoblasts

ObjectiveCold-sensitive ion channels, such as transient receptor potential melastatin (TRPM) 8 and transient receptor potential ankyrin (TRPA) 1, may play a crucial role in the nociceptive function of odontoblasts, whereas expression of these TRP channels in human native odontoblasts remains to be elucidated. This study aimed to analyze the expression of TRPM8 and TRPA1 in freshly isolated native human odontoblasts. DesignOdontoblasts were isolated from freshly extracted healthy human teeth (n=4); after removing the inner pulp tissues from the pulp chambers, odontoblasts remaining on the dentin surface were washed out with phosphate buffered saline and collected. Reverse transcription-polymerase chain reaction was employed to compare the expression levels of TRPM8, TRPA1, and dentin matrix acidic phosphoprotein 1 (DMP1) mRNAs between the isolated odontoblasts and the inner pulp tissues. The isolated cells were subjected to immunolocalization of TRPM8 and nestin. Paraformaldehyde-fixed, EDTA-demineralized frozen sections obtained from freshly extracted healthy human teeth (n=4) were also analyzed immunohistochemically using anti-nestin, TRPM8, and TRPA1 antibodies. ResultsExpression levels of TRPM8 and DMP1 in the isolated odontoblasts were significantly higher than those in the inner pulp tissues (p<0.05). Expression of TRPM8 and nestin was observed in the odontoblastic layer of the dental pulp tissue and isolated odontoblasts, while expression of TRPA1 was not detected. ConclusionsTRPM8, but not TRPA1, was detected in freshly isolated native human odontoblasts at the protein and mRNA levels, suggesting that odontoblasts play an important role in detecting external cold stimulation via TRPM8 in healthy condition.