DOCSLIB.ORG

Combined Targeting of MEK and PI3K/Mtor Effector Pathways Is Necessary to Effectively Inhibit NRAS Mutant Melanoma in Vitro and in Vivo

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Combined Targeting of MEK and PI3K/Mtor Effector Pathways Is Necessary to Effectively Inhibit NRAS Mutant Melanoma in Vitro and in Vivo Combined targeting of MEK and PI3K/mTOR effector pathways is necessary to effectively inhibit NRAS mutant melanoma in vitro and in vivo Christian Poscha,b,1, Homayoun Moslehia, Luzviminda Feeneya, Gary A. Greena, Anoosheh Ebaeea, Valentin Feichtenschlagerb, Kim Chonga, Lily Penga, Michelle T. Dimona, Thomas Phillipsa, Adil I. Daudc, Timothy H. McCalmontd, Philip E. LeBoitd, and Susana Ortiz-Urdaa aDepartment of Dermatology, Mount Zion Cancer Research Center, and dDepartment of Pathology, University of California, San Francisco, CA 94115; bDepartment of Dermatology, Rudolfstiftung Hospital, A-1030 Vienna, Austria; and cDepartment of Dermatology, University of California, San Francisco, CA 94143 Edited* by James E. Cleaver, University of California, San Francisco, CA, and approved January 23, 2013 (received for review September 14, 2012) Activating mutations in the neuroblastoma rat sarcoma viral onco- with metastatic melanoma. The lack of responses among the first gene homolog (NRAS) gene are common genetic events in malig- 14 patients led to the early closure of the trial. A paucity of ef- nant melanoma being found in 15–25% of cases. NRAS is thought to ficacy has also been observed for this approach in other RAS- activate both mitogen activated protein kinase (MAPK) and PI3K mutated malignancies. Recently, an oral mitogen activated protein signaling in melanoma cells. We studied the influence of different (MAP)/extracellular signal-regulated (ERK) kinase (MEK) in- components on the MAP/extracellular signal-regulated (ERK) kinase hibitor (MEK162) was tested in patients with metastatic mela- (MEK) and PI3K/mammalian target of rapamycin (mTOR)-signaling noma harboring murine sarcoma viral oncogene homolog B1 cascade in NRAS mutant melanoma cells. In general, these cells were (BRAF) or NRAS mutations with encouraging results (12). In this more sensitive to MEK inhibition compared with inhibition in the PI3K/ study, we evaluate in detail NRAS mutant primary melanomas, mTOR cascade. Combined targeting of MEK and PI3K was superior to melanoma metastases, and 10 human NRAS mutant melanoma MEK and mTOR inhibition in all NRAS mutant melanoma cell lines cell lines. The expression and role of MEK/ERK and PI3K/ 1,2 mammalian target of rapamycin (mTOR) phospho-proteins in via- tested, suggesting that PI3K signaling is more important for cell survival MEDICAL SCIENCES in NRAS mutant melanoma when MEK is inhibited. However, targeting bility, growth, and therapeutics of NRAS mutant melanoma tumors are assessed. Our data show that combined targeting of MEK and of PI3K/mTOR in combination with MEK inhibitors is necessary to 1,2 PI3K/mTOR is necessary to regress NRAS mutant melanoma, effectively abolish growth of NRAS mutant melanoma cells in vitro 1,2 thus opening the possibility of a beneficial treatment strategy. and regress xenografted NRAS mutant melanoma. Furthermore, we showed that MEK and PI3K/mTOR1,2 inhibition is synergistic. Expression Results analysis confirms that combined MEK and PI3K/mTOR inhibition pre- 1,2 NRAS Mutant Melanoma Activates the MEK/ERK, the PI3K/mTOR dominantly influences genes in the rat sarcoma (RAS) pathway and Pathway, or both. Levels of phospho-ERK, p-MEK phospho- growth factor receptor pathways, which signal through MEK/ERK murine thymoma viral oncogene homolog 1 (p-AKT), phospho- and PI3K/mTOR, respectively. Our results suggest that combined target- S6 ribosomal protein (p-S6), and phosphatase and tensin homolog ing of the MEK/ERK and PI3K/mTOR pathways has antitumor activity (PTEN) were measured in 14 primary melanomas and 18 me- and might serve as a therapeutic option in the treatment of NRAS tastases from 32 patient specimens of NRAS-mutated melanoma. mutant melanoma, for which there are currently no effective therapies. Sample information, including mutation status, is provided in Table S1. Protein levels were measured by immunohisto- ncogenic mutations in codons 12, 13, or 61 of the rat sarcoma chemistry (IHC) and evaluated as the average rating of staining O(RAS) family of small GTPases, Kirsten rat sarcoma viral intensity by four independent reviewers on a scale from 3 =+++ oncogene homolog (KRAS), Harvey rat sarcoma viral oncogene positive to 0 = negative. Analysis of inter- and intrarater re- homolog (HRAS), and neuroblastoma RAS viral oncogene ho- liability can be found in Fig. S1. Across all patients, tumors dis- molog (NRAS) occur in approximately one-third of all human played the strongest staining for p-ERK (1.34 ± 0.14) and p-S6 cancers with NRAS mutations found in about 15–20% of mela- (1.16 ± 0.12). Comparing the staining patterns between nomas (1–7). Mutated RAS proteins activate signaling pathways patients, scoring results were divided into quartiles. Negative that promote the cell division cycle and cell growth and suppress staining was defined as a score ≤0.75 (quartile 1), and positive apoptosis. Small interfering RNA (siRNA)-mediated depletion of staining was defined as a score >0.75 (quartile 2, 3, 4). Positive NRAS in melanoma cell lines inhibits proliferation and renders staining for p-ERK or p-S6 or both was found in 95.24% of cells sensitive to chemotherapy, making mutant NRAS and its samples stained, with 42.86% of patients positive for both pro- signaling effectors relevant targets for melanoma therapy (8, 9). teins, 28.57% positive only for p-MEK, and 23.81% positive only Efforts at developing therapeutics that inhibit mutant RAS di- for p-S6 (Fig. 1). In 28.6% of patient samples, p-ERK/p-MEK rectly have so far not been successful. The high affinity of RAS for staining was negative. This was predominantly the case in mel- GTP and the high concentrations of GTP intracellularly has anoma metastasis although it was not statistically significant; meant that the identification of small molecules, which selectively 57.9% of slides expressed PTEN, with an average staining prevent accumulation of RAS-GTP, has not been possible (10). Targeting mutant NRAS with siRNA is still limited to preclinical fi models because of the signi cant challenge in delivering antisense Author contributions: C.P., A.I.D., T.H.M., P.E.L., and S.O.-U. designed research; C.P., H.M., oligonucleotides in vivo. The response of NRAS mutant mela- L.F., G.A.G., A.E., V.F., K.C., L.P., and T.P. performed research; T.H.M. and P.E.L. contrib- noma and other melanomas to various chemotherapeutic regi- uted new reagents/analytic tools; C.P., G.A.G., A.E., V.F., K.C., L.P., M.T.D., A.I.D., and ments has been very scarce with only 6% of patients responding S.O.-U. analyzed data; and C.P. and S.O.-U. wrote the paper. (11). Alternatively, farnesyltransferase inhibitors (FTIs) were The authors declare no conflict of interest. thought to inhibit RAS activation by blocking farnesylation, a key *This Direct Submission article had a prearranged editor. posttranslational modification step of RAS that is essential for 1To whom correspondence should be addressed. E-mail: [email protected]. RAS function. One FTI, R115777 (also known as tipifarnib), was This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. evaluated in a single-agent, single-arm phase II trial in patients 1073/pnas.1216013110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1216013110 PNAS | March 5, 2013 | vol. 110 | no. 10 | 4015–4020 Downloaded by guest on September 28, 2021 Combination of MEK and PI3K/mTOR1,2 Inhibitors Is Most Effective in Reducing Cell Viability in NRAS Mutant Melanoma. We further ex- amined the growth inhibitory effect of different combinations of selective inhibitors. Combining a MEKi with a PI3Ki, mTORi1, mTORi1,2, or AKTi resulted in greater growth inhibition than with a MEKi alone in all cell lines. The combination of MEKi+PI3Ki was superior to combinations of MEKi with mTORi1,mTORi1,2, or AKTi. However, the strongest decrease in cell viability was seen with MEKi+PI3K/mTORi1,2 (Fig. 3 and Fig. S3). As expected, immunoblot analyses revealed “paradoxical” AKT hyperphosphorylation when cells were treated with the AKTi. This previously described effect is not due to enhance- ment of upstream signals to compensate for AKT signal loss, but rather is related to occupation of the AKT nucleotide-binding pocket by the AKT inhibitor (14,15). We further found that MEKi+mTORi1,2 reduced levels of p-S6 more potently than MEKi+PI3Ki. Interestingly, the latter has a stronger effect in the reduction of viability in NRAS cell lines. Only the combination of MEKi and PI3K/mTORi1,2 showed complete suppression of p-ERK, p-AKT, and p-S6 in D04, MaMel30I, MM485, WM3670, WM3060, and WM1366 and almost complete suppression in Sk-Mel-2 and WM3629 cells (Fig. 3 and Fig. S3). Combined Inhibition of MEK/ERK and PI3K/mTOR Pathways Induces Apoptosis in NRAS Mutant Melanoma Cells. +++ To evaluate if growth Fig. 1. (A) Representative positive staining for p-MEK, p-ERK, p-AKT, and inhibition induced with single or combination treatment is due to p-S6. (B) Representative negative staining for p-MEK, p-ERK, p-AKT, and p-S6. apoptosis, we determined the number of cells with phosphatidyl (C) Staining intensity of proteins in the MEK/ERK and PI3K/mTOR cascade across 32 different NRAS mutant melanomas. Error bars indicate the SD for the serine exposed outside the cell membrane by labeling with an Annexin V-FITC antibody. Cells with loss of membrane integrity, intensity scores from four independent raters. (D) Percentage of patients with fl fi positive and negative staining for p-MEK/p-ERK, p-AKT/p-S6, or both. re ecting the latest stage of cell death, were identi ed by propi- dium iodide (PI) staining. Results confirmed that the reduction of cell count in viability assays is a result of apoptosis (16).
Load more

Recommended publications
  • Deregulated Gene Expression Pathways in Myelodysplastic Syndrome Hematopoietic Stem Cells
    Leukemia (2010) 24, 756–764 & 2010 Macmillan Publishers Limited All rights reserved 0887-6924/10 $32.00 www.nature.com/leu ORIGINAL ARTICLE Deregulated gene expression pathways in myelodysplastic syndrome hematopoietic stem cells A Pellagatti1, M Cazzola2, A Giagounidis3, J Perry1, L Malcovati2, MG Della Porta2,MJa¨dersten4, S Killick5, A Verma6, CJ Norbury7, E Hellstro¨m-Lindberg4, JS Wainscoat1 and J Boultwood1 1LRF Molecular Haematology Unit, NDCLS, John Radcliffe Hospital, Oxford, UK; 2Department of Hematology Oncology, University of Pavia Medical School, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy; 3Medizinische Klinik II, St Johannes Hospital, Duisburg, Germany; 4Division of Hematology, Department of Medicine, Karolinska Institutet, Stockholm, Sweden; 5Department of Haematology, Royal Bournemouth Hospital, Bournemouth, UK; 6Albert Einstein College of Medicine, Bronx, NY, USA and 7Sir William Dunn School of Pathology, University of Oxford, Oxford, UK To gain insight into the molecular pathogenesis of the the World Health Organization.6,7 Patients with refractory myelodysplastic syndromes (MDS), we performed global gene anemia (RA) with or without ringed sideroblasts, according to expression profiling and pathway analysis on the hemato- poietic stem cells (HSC) of 183 MDS patients as compared with the the French–American–British classification, were subdivided HSC of 17 healthy controls. The most significantly deregulated based on the presence or absence of multilineage dysplasia. In pathways in MDS include interferon signaling, thrombopoietin addition, patients with RA with excess blasts (RAEB) were signaling and the Wnt pathways. Among the most signifi- subdivided into two categories, RAEB1 and RAEB2, based on the cantly deregulated gene pathways in early MDS are immuno- percentage of bone marrow blasts.
    [Show full text]
  • Outlier Kinase Expression by RNA Sequencing As Targets for Precision Therapy
    Published OnlineFirst February 5, 2013; DOI: 10.1158/2159-8290.CD-12-0336 RESEARCH ARTICLE Outlier Kinase Expression by RNA Sequencing as Targets for Precision Therapy Vishal Kothari 1 , Iris Wei 2 , Sunita Shankar 1 , 3 , Shanker Kalyana-Sundaram 1 , 3 , 8 , Lidong Wang 2 , Linda W. Ma 1 , Pankaj Vats 1 , Catherine S. Grasso 1 , Dan R. Robinson 1 , 3 , Yi-Mi Wu 1 , 3 , Xuhong Cao 7 , Diane M. Simeone 2 , 4 , 5 , Arul M. Chinnaiyan 1 , 3 , 4 , 6 , 7 , and Chandan Kumar-Sinha 1 , 3 ABSTRACT Protein kinases represent the most effective class of therapeutic targets in cancer; therefore, determination of kinase aberrations is a major focus of cancer genomic studies. Here, we analyzed transcriptome sequencing data from a compendium of 482 cancer and benign samples from 25 different tissue types, and defi ned distinct “outlier kinases” in individual breast and pancreatic cancer samples, based on highest levels of absolute and differential expression. Frequent outlier kinases in breast cancer included therapeutic targets like ERBB2 and FGFR4 , distinct from MET , AKT2 , and PLK2 in pancreatic cancer. Outlier kinases imparted sample-specifi c depend- encies in various cell lines, as tested by siRNA knockdown and/or pharmacologic inhibition. Outlier expression of polo-like kinases was observed in a subset of KRAS -dependent pancreatic cancer cell lines, and conferred increased sensitivity to the pan-PLK inhibitor BI-6727. Our results suggest that outlier kinases represent effective precision therapeutic targets that are readily identifi able through RNA sequencing of tumors. SIGNIFICANCE: Various breast and pancreatic cancer cell lines display sensitivity to knockdown or pharmacologic inhibition of sample-specifi c outlier kinases identifi ed by high-throughput transcrip- tome sequencing.
    [Show full text]
  • Systematic Screening for Potential Therapeutic Targets in Osteosarcoma Through a Kinome-Wide CRISPR-Cas9 Library
    Cancer Biol Med 2020. doi: 10.20892/j.issn.2095-3941.2020.0162 ORIGINAL ARTICLE Systematic screening for potential therapeutic targets in osteosarcoma through a kinome-wide CRISPR-Cas9 library Yuanzhong Wu*, Liwen Zhou*, Zifeng Wang, Xin Wang, Ruhua Zhang, Lisi Zheng, Tiebang Kang Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou 510060, China ABSTRACT Objective: Osteosarcoma is the most common primary malignant bone tumor. However, the survival of patients with osteosarcoma has remained unchanged during the past 30 years, owing to a lack of efficient therapeutic targets. Methods: We constructed a kinome-targeting CRISPR-Cas9 library containing 507 kinases and 100 nontargeting controls and screened the potential kinase targets in osteosarcoma. The CRISPR screening sequencing data were analyzed with the Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK) Python package. The functional data were applied in the 143B cell line through lenti-CRISPR-mediated gene knockout. The clinical significance of kinases in the survival of patients with osteosarcoma was analyzed in the R2: Genomics Analysis and Visualization Platform. Results: We identified 53 potential kinase targets in osteosarcoma. Among these targets, we analyzed 3 kinases, TRRAP, PKMYT1, and TP53RK, to validate their oncogenic functions in osteosarcoma. PKMYT1 and TP53RK showed higher expression in osteosarcoma than in normal bone tissue, whereas TRRAP showed no significant difference. High expression of all 3 kinases was associated with relatively poor prognosis in patients with osteosarcoma. Conclusions: Our results not only offer potential therapeutic kinase targets in osteosarcoma but also provide a paradigm for functional genetic screening by using a CRISPR-Cas9 library, including target design, library construction, screening workflow, data analysis, and functional validation.
    [Show full text]
  • Advancing a Clinically Relevant Perspective of the Clonal Nature of Cancer
    Advancing a clinically relevant perspective of the clonal nature of cancer Christian Ruiza,b, Elizabeth Lenkiewicza, Lisa Eversa, Tara Holleya, Alex Robesona, Jeffrey Kieferc, Michael J. Demeurea,d, Michael A. Hollingsworthe, Michael Shenf, Donna Prunkardf, Peter S. Rabinovitchf, Tobias Zellwegerg, Spyro Moussesc, Jeffrey M. Trenta,h, John D. Carpteni, Lukas Bubendorfb, Daniel Von Hoffa,d, and Michael T. Barretta,1 aClinical Translational Research Division, Translational Genomics Research Institute, Scottsdale, AZ 85259; bInstitute for Pathology, University Hospital Basel, University of Basel, 4031 Basel, Switzerland; cGenetic Basis of Human Disease, Translational Genomics Research Institute, Phoenix, AZ 85004; dVirginia G. Piper Cancer Center, Scottsdale Healthcare, Scottsdale, AZ 85258; eEppley Institute for Research in Cancer and Allied Diseases, Nebraska Medical Center, Omaha, NE 68198; fDepartment of Pathology, University of Washington, Seattle, WA 98105; gDivision of Urology, St. Claraspital and University of Basel, 4058 Basel, Switzerland; hVan Andel Research Institute, Grand Rapids, MI 49503; and iIntegrated Cancer Genomics Division, Translational Genomics Research Institute, Phoenix, AZ 85004 Edited* by George F. Vande Woude, Van Andel Research Institute, Grand Rapids, MI, and approved June 10, 2011 (received for review March 11, 2011) Cancers frequently arise as a result of an acquired genomic insta- on the basis of morphology alone (8). Thus, the application of bility and the subsequent clonal evolution of neoplastic cells with purification methods such as laser capture microdissection does variable patterns of genetic aberrations. Thus, the presence and not resolve the complexities of many samples. A second approach behaviors of distinct clonal populations in each patient’s tumor is to passage tumor biopsies in tissue culture or in xenografts (4, 9– may underlie multiple clinical phenotypes in cancers.
    [Show full text]
  • Circrna LRIG3 Knockdown Inhibits Hepatocellular Carcinoma Progression by Regulating Mir-223-3P and MAPK/ERK Pathway
    CircRNA LRIG3 knockdown inhibits hepatocellular carcinoma progression by regulating miR-223-3p and MAPK/ERK pathway Type Research paper Keywords hepatocellular carcinoma, miR-223-3p, circ_LRIG3, MAP2K6, MAPK/ERK pathway Abstract Introduction Emerging evidence suggests that circular RNAs (circRNAs) play critical roles in tumorigenesis. However, the roles and molecular mechanisms of circRNA leucine-rich repeat immunoglobulin domain-containing protein 3 (circ_LRIG3) in hepatocellular carcinoma (HCC) has not been investigated. Material and methods The expression levels of circ_LRIG3, miR-223-3p, and mitogen-activated protein kinase kinase 6 (MAP2K6) were determined by qRT-PCR. Flow cytometry was applied to determine the cell cycle distribution and apoptosis. Cell proliferation, migration and invasion were assessed by MTT, colony formation, and transwell assays. Western blot assay was employed to measure the protein levels of the snail, E-cadherin, MAP2K6, mitogen-activated protein kinase (MAPK), phospho-MAPK (p- MAPK), extracellular signal-regulated kinases (ERKs), and phospho-ERKs (p- ERKs). The relationship between miR-223-3p and circ_LRIG3 or MAP2K6 was predicted by bioinformatics tools and verified by dual-luciferase reporter assay. A xenograft tumor model was established to confirm the functions of circ_LRIG3 in vivo. Results Preprint Circ_LRIG3 and MAP2K6 expression were enhanced while miR-223-3p abundance was reduced in HCC tissues and cells. Knockdown of circ_LRIG3 inhibited cell proliferation, metastasis, and increasing apoptosis. MiR-223-3p was a target of circ_LRIG3, and its downregulation reversed the inhibitory effect of circ_LRIG3 knockdown on the progression of HCC cells. Moreover, MAP2K6 could bind to miR-223-3p, and MAP2K6 upregulation also abolished the suppressive impact of circ_LRIG3 interference on progression of HCC cells.
    [Show full text]
  • Profiling Data
    Compound Name DiscoveRx Gene Symbol Entrez Gene Percent Compound Symbol Control Concentration (nM) JNK-IN-8 AAK1 AAK1 69 1000 JNK-IN-8 ABL1(E255K)-phosphorylated ABL1 100 1000 JNK-IN-8 ABL1(F317I)-nonphosphorylated ABL1 87 1000 JNK-IN-8 ABL1(F317I)-phosphorylated ABL1 100 1000 JNK-IN-8 ABL1(F317L)-nonphosphorylated ABL1 65 1000 JNK-IN-8 ABL1(F317L)-phosphorylated ABL1 61 1000 JNK-IN-8 ABL1(H396P)-nonphosphorylated ABL1 42 1000 JNK-IN-8 ABL1(H396P)-phosphorylated ABL1 60 1000 JNK-IN-8 ABL1(M351T)-phosphorylated ABL1 81 1000 JNK-IN-8 ABL1(Q252H)-nonphosphorylated ABL1 100 1000 JNK-IN-8 ABL1(Q252H)-phosphorylated ABL1 56 1000 JNK-IN-8 ABL1(T315I)-nonphosphorylated ABL1 100 1000 JNK-IN-8 ABL1(T315I)-phosphorylated ABL1 92 1000 JNK-IN-8 ABL1(Y253F)-phosphorylated ABL1 71 1000 JNK-IN-8 ABL1-nonphosphorylated ABL1 97 1000 JNK-IN-8 ABL1-phosphorylated ABL1 100 1000 JNK-IN-8 ABL2 ABL2 97 1000 JNK-IN-8 ACVR1 ACVR1 100 1000 JNK-IN-8 ACVR1B ACVR1B 88 1000 JNK-IN-8 ACVR2A ACVR2A 100 1000 JNK-IN-8 ACVR2B ACVR2B 100 1000 JNK-IN-8 ACVRL1 ACVRL1 96 1000 JNK-IN-8 ADCK3 CABC1 100 1000 JNK-IN-8 ADCK4 ADCK4 93 1000 JNK-IN-8 AKT1 AKT1 100 1000 JNK-IN-8 AKT2 AKT2 100 1000 JNK-IN-8 AKT3 AKT3 100 1000 JNK-IN-8 ALK ALK 85 1000 JNK-IN-8 AMPK-alpha1 PRKAA1 100 1000 JNK-IN-8 AMPK-alpha2 PRKAA2 84 1000 JNK-IN-8 ANKK1 ANKK1 75 1000 JNK-IN-8 ARK5 NUAK1 100 1000 JNK-IN-8 ASK1 MAP3K5 100 1000 JNK-IN-8 ASK2 MAP3K6 93 1000 JNK-IN-8 AURKA AURKA 100 1000 JNK-IN-8 AURKA AURKA 84 1000 JNK-IN-8 AURKB AURKB 83 1000 JNK-IN-8 AURKB AURKB 96 1000 JNK-IN-8 AURKC AURKC 95 1000 JNK-IN-8
    [Show full text]
  • The Number of Genes
    Table S1. The numbers of KD genes in each KD time The number The number The number The number Cell lines of genes of genes of genes of genes (96h) (120h) (144h) PC3 3980 3822 128 1725 A549 3724 3724 0 0 MCF7 3688 3471 0 1837 HT29 3665 3665 0 0 A375 3826 3826 0 0 HA1E 3801 3801 0 0 VCAP 4134 34 4121 0 HCC515 3522 3522 0 0 Table S2. The predicted results in the PC3 cell line on the LINCS II data id target rank A07563059 ADRB2 48 A12896037 ADRA2C 91 A13021932 YES1 77 PPM1B;PPP1CC;PPP2CA; A13254067 584;1326;297;171;3335 PTPN1;PPP2R5A A16347691 GMNN 2219 PIK3CB;MTOR;PIK3CA;PIK A28467416 18;10;9;13;8 3CG;PIK3CD A28545468 EHMT2;MAOB 14;67 A29520968 HSPB1 1770 A48881734 EZH2 1596 A52922642 CACNA1C 201 A64553394 ADRB2 155 A65730376 DOT1L 3764 A82035391 JUN 378 A82156122 DPP4 771 HRH1;HTR2C;CHRM3;CH A82772293 2756;2354;2808;2367 RM1 A86248581 CDA 1785 A92800748 TEK 459 A93093700 LMNA 1399 K00152668 RARB 105 K01577834 ADORA2A 525 K01674964 HRH1;BLM 31;1314 K02314383 AR 132 K03194791 PDE4D 30 K03390685 MAP2K1 77 K06762493 GMNN;APEX1 1523;2360 K07106112 ERBB4;ERBB2;EGFR 497;60;23 K07310275 AKT1;MTOR;PIK3CA 13;12;1 K07753030 RGS4;BLM 3736;3080 K08109215 BRD2;BRD3;BRD4 1413;2786;3 K08248804 XIAP 88 K08586861 TBXA2R;MBNL1 297;3428 K08832567 GMNN;CA12 2544;50 LMNA;NFKB1;APEX1;EH K08976401 1322;341;3206;123 MT2 K09372874 IMPDH2 232 K09711437 PLA2G2A 59 K10859802 GPR119 214 K11267252 RET;ALK 395;760 K12609457 LMNA 907 K13094524 BRD4 7 K13662825 CDK4;CDK9;CDK5;CDK1 34;58;13;18 K14704277 LMNA;BLM 1697;1238 K14870255 AXL 1696 K15170068 MAN2B1 1756 K15179879
    [Show full text]
  • PRKACA Mediates Resistance to HER2-Targeted Therapy in Breast Cancer Cells and Restores Anti-Apoptotic Signaling
    Oncogene (2015) 34, 2061–2071 © 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc ORIGINAL ARTICLE PRKACA mediates resistance to HER2-targeted therapy in breast cancer cells and restores anti-apoptotic signaling SE Moody1,2,3, AC Schinzel1, S Singh1, F Izzo1, MR Strickland1, L Luo1,2, SR Thomas3, JS Boehm3, SY Kim4, ZC Wang5,6 and WC Hahn1,2,3 Targeting HER2 with antibodies or small molecule inhibitors in HER2-positive breast cancer leads to improved survival, but resistance is a common clinical problem. To uncover novel mechanisms of resistance to anti-HER2 therapy in breast cancer, we performed a kinase open reading frame screen to identify genes that rescue HER2-amplified breast cancer cells from HER2 inhibition or suppression. In addition to multiple members of the MAPK (mitogen-activated protein kinase) and PI3K (phosphoinositide 3-kinase) signaling pathways, we discovered that expression of the survival kinases PRKACA and PIM1 rescued cells from anti-HER2 therapy. Furthermore, we observed elevated PRKACA expression in trastuzumab-resistant breast cancer samples, indicating that this pathway is activated in breast cancers that are clinically resistant to trastuzumab-containing therapy. We found that neither PRKACA nor PIM1 restored MAPK or PI3K activation after lapatinib or trastuzumab treatment, but rather inactivated the pro-apoptotic protein BAD, the BCl-2-associated death promoter, thereby permitting survival signaling through BCL- XL. Pharmacological blockade of BCL-XL/BCL-2 partially abrogated the rescue effects conferred by PRKACA and PIM1, and sensitized cells to lapatinib treatment. These observations suggest that combined targeting of HER2 and the BCL-XL/BCL-2 anti-apoptotic pathway may increase responses to anti-HER2 therapy in breast cancer and decrease the emergence of resistant disease.
    [Show full text]
  • PRODUCT SPECIFICATION Anti-MAP3K10 Product Datasheet
    Anti-MAP3K10 Product Datasheet Polyclonal Antibody PRODUCT SPECIFICATION Product Name Anti-MAP3K10 Product Number HPA007039 Gene Description mitogen-activated protein kinase kinase kinase 10 Clonality Polyclonal Isotype IgG Host Rabbit Antigen Sequence Recombinant Protein Epitope Signature Tag (PrEST) antigen sequence: LPSGFEHKITVQASPTLDKRKGSDGASPPASPSIIPRLRAIRLTPVDCGG SSSGSSSGGSGTWSRGGPPKKEELVGGKKKGRTWGPSSTLQKERVGGEER LKGLGEGSKQWSSS Purification Method Affinity purified using the PrEST antigen as affinity ligand Verified Species Human Reactivity Recommended IHC (Immunohistochemistry) Applications - Antibody dilution: 1:200 - 1:500 - Retrieval method: HIER pH6 ICC-IF (Immunofluorescence) - Fixation/Permeabilization: PFA/Triton X-100 - Working concentration: 0.25-2 µg/ml Characterization Data Available at atlasantibodies.com/products/HPA007039 Buffer 40% glycerol and PBS (pH 7.2). 0.02% sodium azide is added as preservative. Concentration Lot dependent Storage Store at +4°C for short term storage. Long time storage is recommended at -20°C. Notes Gently mix before use. Optimal concentrations and conditions for each application should be determined by the user. For protocols, additional product information, such as images and references, see atlasantibodies.com. Product of Sweden. For research use only. Not intended for pharmaceutical development, diagnostic, therapeutic or any in vivo use. No products from Atlas Antibodies may be resold, modified for resale or used to manufacture commercial products without prior written approval from Atlas Antibodies AB. Warranty: The products supplied by Atlas Antibodies are warranted to meet stated product specifications and to conform to label descriptions when used and stored properly. Unless otherwise stated, this warranty is limited to one year from date of sales for products used, handled and stored according to Atlas Antibodies AB's instructions. Atlas Antibodies AB's sole liability is limited to replacement of the product or refund of the purchase price.
    [Show full text]
  • Circrna-006258 Sponge-Adsorbs Mir-574-5P to Regulate Cell Growth and Milk Synthesis Via EVI5L in Goat Mammary Epithelial Cells
    G C A T T A C G G C A T genes Article CircRNA-006258 Sponge-Adsorbs miR-574-5p to Regulate Cell Growth and Milk Synthesis via EVI5L in Goat Mammary Epithelial Cells Meng Zhang y , Li Ma y, Yuhan Liu, Yonglong He, Guang Li, Xiaopeng An and Binyun Cao * College of Animal Science and Technology, Northwest A&F University, Yangling 712100, Shanxi, China; [email protected] (M.Z.); [email protected] (L.M.); [email protected] (Y.L.); [email protected] (Y.H.); [email protected] (G.L.); [email protected] (X.A.) * Correspondence: [email protected]; Tel.: +86-29-87092102 These authors contributed equally to this work. y Received: 28 May 2020; Accepted: 25 June 2020; Published: 28 June 2020 Abstract: The development of the udder and the milk yield are closely related to the number and vitality of mammary epithelial cells. Many previous studies have proved that non-coding RNAs (ncRNAs) are widely involved in mammary gland development and the physiological activities of lactation. Our laboratory previous sequencing data revealed that miR-574-5p was differentially expressed during the colostrum and peak lactation stages, while the molecular mechanism of the regulatory effect of miR-574-5p on goat mammary epithelial cells (GMECs) is unclear. In this study, the targeting relationship was detected between miR-574-5p or ecotropic viral integration site 5-like (EVI5L) and circRNA-006258. The results declared that miR-574-5p induced the down-regulation of EVI5L expression at both the mRNA and protein levels, while circRNA-006258 relieved the inhibitory effect through adsorbing miR-574-5p.
    [Show full text]
  • Supplemental Material 1
    Cdk20 Csrp2bp Bub1 Tmed3 1700012B15Rik Uck1 Atad2 Pbk Rfc4 Ucp3 Mtmr7 Bid Rad9 Hadh Zc3h12a Bcl2l11 1700052K11Rik Cd83 Ncaph Cdca7l Ezh2 Kif23 Rel Cdh13 Nr4a1 Pola1 Phf19 Vldlr Snx7 Fam96a Ripk2 Ccne2 Ncapd2 Pqlc3 A130009I22Rik Niacr1 Peli1 Cenpn Ralgds Dbf4 Trpc6 Gadd45b Dyrk2 Il1b Prim1 Pcna Dnmt1 Ttc30a1 Cflar Fas Tro Tank Rapgef2 Icosl 9930012K11Rik Cd28 Ccdc86 Ube2f Klhl6 Commd8 Ccrl2 D4Wsu53e Tnfaip8l2 Ehd1 Kdm2b Uhrf1 Trim37 Tecrl AI467606 Ilf2 Cobra1 Aurkb Cdc42ep2 Nfkbil2 Appbp2 Nfkbiz Zfp85-rs1 Nlrp3 Zmym5 Il6 Foxo3 Sept3 Ets2 Serpinb2 Smc3 Msh6 Itga5 Gpsm3 Slbp Dennd4c Rffl Trappc1 Mrpl34 Socs3 Ocel1 Tnfaip3 Swap70 1110003O08Rik Junb Pmm1 Cxcl10 Brca1 Acpl2 Coq10b Marcksl1 Map3k10 Braf Tnf Icam1 Pold3 AI462493 Nfkbia Dnmbp Dnajc9 Tnip1 Chfr Trim13 Slc35c2 Rb1 C78513 Limk1 C1qa Fen1 BC088983 Tsc22d3 Tnfsf9 Mapk3 E2f1 Zc3h12c Casp4 Ttf2 Maff Gmnn Zfp715 Mdm2 Mcm7 Irf1 Parp1 Ftsj2 Myo10 Irak2 Mid1ip1 Apeh Rad51c Map3k4 Tk1 E2f7 Tiparp Ptgs2 Spata13 Hmga2-ps1 Wdhd1 Clspn Ubac1 Erp29 Thap4 Dna2 Prkag2 Il1a Rfwd3 Gbp3 Cdca5 3110043O21Rik Clec4e Cxcl11 Speg Tarbp2 Cdk2 Zufsp Cdca2 E2f2 1700054N08Rik Tnfsf10 5730499H23Rik Inf2 C1qb Anapc5 Sco1 Bst2 2010107G23Rik Cnpy4 D10Wsu102e Brip1 H2-D1 Gpd1l Suv420h2 4930579G24Rik Cdc6 Cntnap2 Cyp2b10 Plod2 Irf7 C2 Lsm3 9330175E14Rik Aak1 Ick Rassf7 Cd22 Cd72 Tap1 Tcn2 Malt1 Uba7 Atp1a2 Ar Tap2 Cd86 Herc5 Heca Atp2a2 Ttc28 Dhrs3 Stat1 Ccdc22 Abca5 Nhlrc2 Ddx58 Gpx1 Dgkd Fbxl7 Lap3 Lama4 Neil1 Enpp4 Tpst1 Cybb Ccne1 Sec24d Fcgr4 Hk3 Rasgrp2 Dcun1d2 Vegfa Ecsit Ddx60 Eif2ak2 Cyp2r1 Gimap4 Fip1l1
    [Show full text]
  • Novel Functions of Death-Associated Protein Kinases Through Mitogen-Activated Protein Kinase-Related Signals
    International Journal of Molecular Sciences Article Novel Functions of Death-Associated Protein Kinases through Mitogen-Activated Protein Kinase-Related Signals Mohamed Elbadawy 1,2,† , Tatsuya Usui 1,*,†, Hideyuki Yamawaki 3 and Kazuaki Sasaki 1 1 Laboratory of Veterinary Pharmacology, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan; [email protected] (M.E.); [email protected] (K.S.) 2 Department of Pharmacology, Faculty of Veterinary Medicine, Benha University, Moshtohor, Elqaliobiya, Toukh 13736, Egypt 3 Laboratory of Veterinary Pharmacology, School of Veterinary Medicine, Kitasato University, Towada, Aomori 034-8628, Japan; [email protected] * Correspondence: [email protected]; Tel./Fax: +81-42-367-5769 † These authors contributed equally to this work. Received: 13 September 2018; Accepted: 1 October 2018; Published: 4 October 2018 Abstract: Death associated protein kinase (DAPK) is a calcium/calmodulin-regulated serine/threonine kinase; its main function is to regulate cell death. DAPK family proteins consist of DAPK1, DAPK2, DAPK3, DAPK-related apoptosis-inducing protein kinases (DRAK)-1 and DRAK-2. In this review, we discuss the roles and regulatory mechanisms of DAPK family members and their relevance to diseases. Furthermore, a special focus is given to several reports describing cross-talks between DAPKs and mitogen-activated protein kinases (MAPK) family members in various pathologies. We also discuss small molecule inhibitors of DAPKs and their potential as therapeutic targets against human diseases. Keywords: MAPK; DAPK; ERK; p38; JNK 1. Introduction: DAPKs, MAPKs Death-associated protein kinase (DAPK) family proteins are closely related, Ca2+/calmodulin (CaM)-regulated serine/threonine kinases, whose members not only possess significant homology in their catalytic domains but also share cell death-associated functions [1,2].
    [Show full text]