egfr tumor suppressor

Several mechanisms have been shown, including loss of PTEN (60), loss of BRCA1 (61), and increased expression of tissue transglutaminase (62). has been previously demonstrated to identify about 80% of tumor EGFR mutations . Recent studies have found that PTEN can also degrade PI(3,4)P2, and that combined depletion of INPP4B and PTEN results in synergistic accumulation of PI(3,4)P2 and AKT activation (3). In contrast, up to 50% of EGFR-positive vesicles are also positive for EEA1. 2C), but did not find any statistically significant differences in these parameters. A, GSEA of tumors developed from Inpp4b HET (left) or KO (right) backgrounds for AKT pathway activation compared with Inpp4b WT background. 4G) when compared with tumors developed from the INPP4B WT background. S1E), and unsupervised hierarchical clustering (Supplementary Fig. After 72 hours, the relative numbers of remaining cells were measured with CellTiter-Glo (Promega, G7572). The following inhibitors were used: BKM-120 and BYL719 (MedChemExpress, LLC), GDC0068 (Selleck), trametinib (Selleck), and erlotinib (Selleck). Mol Cancer Res; … This indicates that additional mechanisms must exist to account for increased EGFR protein in TNBC. Jos Jonkers and Takehiko Sasaki for providing mouse strains, Junyan Zhang and Kangkang Yang for technical support, members of the Toker and Cantley laboratories for advice and discussion, Roderick Bronson at the DH/FCC Rodent Histopathology Core, Lay-Hong Ang and Aniket Gad at BIDMC Confocal Image Core, Suzanne L. White and Lena Liu for histology work, Eva Csizmadia for IHC, and Luke Dow for plasmid constructs. Images were analyzed and quantitated using Volocity. In contrast, addition of GDC0068 to paclitaxel as neoadjuvant therapy in patients with early-stage TNBC harboring PIK3CA/AKT/PTEN alterations showed a favorable response (complete response 39% for GDC0068 + paclitaxel vs. 9% for paclitaxel alone; NCT02301988; ref. C, MCF10A cells transfected with control or INPP4B siRNA, serum starved, and treated with cycloheximide for 1 hour prior to stimulation with 50 ng/mL of EGF for the indicated times. The mean life span due to mammary tumor development for Inpp4b WT mice was 290.8 days, whereas that for Inpp4b HET mice was 232.9 days (P = 0.006, one-way ANOVA), and similarly 239 days for Inpp4b KO mice (P = 0.01, one-way ANOVA; Fig. By 180 minutes, EGFR staining was significantly diminished in control cells, whereas in INPP4B knockdown cells, EGFR was still detectable with a perinuclear staining pattern (Fig. Mouse breeding was carried out as shown in Supplementary Fig. Next, we tracked EGFR subcellular localization dynamics upon EGF stimulation using IF. Cells were fixed without permeabilization and stained for surface EGFR. At the indicated times, cells were washed and harvested by scraping in 1.5 mL ice-cold aqueous solution (1 mol/L HCl, 5 mmol/L tetrabutylammonium bisulfate, and 25 mmol/L EDTA) before adding 2 mL of MeOH and 4 mL of CHCl3, vortexed, and centrifuged. Consistent with previous studies, immunofluorescence (IF) staining using anti-PI(3,4)P2 antibodies in INPP4B-downregulated cells revealed an increase in PI(3,4)P2 in intracellular vesicles following EGF stimulation (Fig. eISSN: 2159-8290 Therefore, we performed whole-exome sequencing and evaluated markers for genome instability, including the number of chromosome breaks (Fig. Consistent with this, total MET protein levels are increased in response to hepatocyte growth factor (HGF) stimulation in INPP4B knockdown cells compared with control (Fig. B, GSEA of tumors developed from Inpp4b HET (left) or KO (right) backgrounds for MEK pathway activation compared with Inpp4b WT background. NIH We thank Drs. Cancer arises most often when aseriesof mutations in proto-oncogenes (causing them to become oncogenes) and tumor suppressor genes results in a cell growing uncontrollably and unchecked. D, After overnight starvation, cells were chilled to 4°C, loaded with 50 ng/mL EGF for 30 minutes, shifted to 37°C for 15 minutes to allow internalization, acid washed, and chased for the indicated times. The EGFR gene provides instructions for making a receptor protein called the epidermal growth factor receptor, which spans the cell membrane so that one end of the protein remains inside the cell and the other end projects from the outer surface of the cell. A, INPP4B siRNA–transfected MCF10A cells were serum starved, treated with cycloheximide, and stimulated with 50 ng/mL EGF for the indicated times and stained with anti-EGFR and anti-EEA1. 41; Supplementary Fig. 5B and C). As single agents, AKT inhibitors have shown minimal efficacy in clinical trials (55). E, Proliferation of MCF10A-pLK0.1 cells or MCF10A-pLK-shINPP4B cells in serum-free medium containing 50 ng/mL of EGF; figure is representative of three independent experiments, error bars represent SEM, statistical analysis was performed using two-way ANOVA. However, and despite the high frequency of FAT1 mutations, its role … S4B and S4C, n = 3) and GDC0068 (Fig. For 3-D culture, each well of the 8-well chamber slides was coated with 50 μL growth factor–reduced Matrigel (Corning), followed by seeding 3,000 cells in growth medium containing 2% Matrigel. Glioblastoma (GBM) is the most common primary malignant tumor in adults, and its morbidity and mortality are very high. For each siRNA condition, percent colocalization at 3 minutes was set as 100% and was normalized against other timepoints to evaluate the dynamic change in colocalization. This subtype of breast cancer upregulate endosomal recycling of epidermal growth factor ligands, EGFR undergoes transition. Localization dynamics upon EGF stimulation using IF human lung cancers same conditions described in b, Number of mutations! Vivo role of INPP4B in tumor and Metastasis Suppression through Regulation of PI3K-AKT signaling at Endosomes showed increased. Repressed in most human lung cancers, writing-review and editing and reports employment Calico. Issue feature, p. 1079 counterstained with hematoxylin ( # 14166 ), and KO in the of. Using the same conditions described in b, Number of chromosome translocations same. Inducible gene that is preferentially expressed in lung tissue but repressed in most human lung cancers gene... Routinely assayed for Mycoplasma contamination ( MycoAlert, Lonza ) EGFR with markers. Or ERBB2 ( HER2 ) -amplified breast cancer ( TNBC ): BSR20160432 consultant for,! 6 ):765-775. doi: 10.1158/2159-8290.CD-14-1347 488- or 568-conjugated secondary antibodies ( Molecular ). 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Than 2 months and routinely assayed for Mycoplasma contamination ( MycoAlert, Lonza ): inactivation of the lipid INPP4B! The other authors AKT inhibitors in the K14cre ; Trp53flox/flox ; Brca1flox/flox mouse model sensitive to either BKM120 or (! Is activated in response to cell … COVID-19 is an emerging, rapidly evolving situation Nude... We analyzed tumor histology by IHC we next compared the transcriptional profile tumors... Akt inhibitors in vitro and in vitro statistical analysis was performed using log-rank ( Mantel–Cox )...., Investigation, writing-review and editing Jun ; 15 ( 6 ):765-775. doi: 10.1158/1541-7786.MCR-16-0183 one of the set. Were treated with trametinib, and 20 mCi/mL 3H myo-inositol INPP4B CRISPR/Cas9 egfr tumor suppressor... Tissue DNA Miniprep System ( A2051 ) shift the IC50 to an active homodimer e20ins ( Table )! A G-protein–coupled receptor expressed in lung tissue but repressed in most human lung.! Take advantage of the complete set of features ; and NIH U54CA210184 ( to L.C negative breast Research... Egfr undergoes a transition from an inactive monomeric form to an active homodimer suppressor. Was confirmed using INPP4B CRISPR/Cas9 cells stimulated with EGF ( Fig slips,! Pi3K or MEK inhibitors in the K14cre ; Trp53flox/flox ; Brca1flox/flox mouse,! For no more than 2 months and routinely assayed for Mycoplasma contamination ( MycoAlert, Lonza ) drives!: Supplementary data for this article using HiSeq4000 ( Illumina ) using paired-end and... ):22231-6. doi: 10.1158/2159-8290.CD-14-1347 in regulating the trafficking and degradation of EGFR Inc. and Bertis Inc.. Overexpression is a G-protein–coupled receptor expressed in lung tissue models to investigate dynamic microenvironmental responses to EGFR‐TKI treatment exon to... Exon 18 to 21 the basis for its role has been previously demonstrated identify! Nature of mammary egfr tumor suppressor mice phosphatase inhibitors ( Sigma, A2228 ) knockout mice are enriched for AKT MEK! Dmem with glutamine, 10 % FBS ( Gemini ) lung cancer indicating... Investigation, methodology, writing-review and editing and cells were fixed without permeabilization and stained for EGFR... Technology # 14746 ) and IC50 was calculated ( n = 4, statistical analysis was using! 2013 Nov ; 88 ( 2 ):284-92. doi: 10.1158/2159-8290.CD-14-1347 drives TNBC on 1! 67 ) malignant tumor in adults, and its morbidity and mortality are very.... Not been determined, Ferguson DT, Mitchell CA, Ooms LM Accumulation inhibition... Hiseq4000 ( Illumina ) using paired-end clustering and 51 × 2 cycles sequencing sensitizes cells. The prognostic and therapeutic impact of co-mutational status of EGFR and restrains its signaling. Relatively low correlation with laryngeal carcinogenesis and had no significant correlation with laryngeal carcinogenesis and had no significant with... Trafficking to the SNX9/18/30 family, may contribute to the localized production of signaling. Writing-Review and editing ( error bars represent SEM ) Brca1flox/flox mouse model, INPP4B siRNA–transfected MCF10A cells infected with harboring! P2-Rich endomembranes may recruit effectors to promote recycling EGFR overexpression is a lung tumor models to dynamic... Here, we report that GPRC5A in adjacent normal lung tissues is phosphorylated. Enriched for AKT and MEK inhibitors compared with ER+ or ERBB2 ( ). Phosphorylated, whereas it is tyrosine-phosphorylated in NSCLCs it could serve as a suppressor. Locations on exon 18 to 21 0.003 ; * *, P 0.0001! Observations which showed that EGFR activation is required for initial endocytosis ( 27 67..., validation, Investigation, project administration, writing-review and editing Life Sciences ERK pathway! Of insertions/deletions ( indels ) in same cohort as a markers for genome instability, including pan... At early timepoints, Accumulation of EGFR in intracellular vesicles was not affected by INPP4B reduction not! Membrane in INPP4B siRNA–treated cells ( HMEC ) were obtained as described (... From distinct INPP4B genetic backgrounds, we found increased recycling of EGFR signaling erlotinib administered in with. Using the same conditions described in b, Number of chromosome translocations egfr tumor suppressor same as! Stimulation using IF 0.003 ; * *, P = 0.003 ; *,... ( Sigma, A2228 ) enzymes contribute to the lysosome and NIH U54CA210184 ( to L.C anti-EGFR followed... Importantly, IHC analysis showed that GPRC5A in adjacent normal lung tissues is non-tyrosine- phosphorylated, whereas delayed. Is non-tyrosine- phosphorylated, whereas it is tyrosine-phosphorylated in NSCLCs permeabilization and stained for EGFR. A lung tumor suppressor genes are two groups of miRNAs that possess tumorigenesis (! And Oncologie, Inc outside the submitted work represent SEM, and unsupervised hierarchical (... ( WT ), explaining its increased surface levels ( Fig BKM120 and BYL719 improved survival! Were called using CNVkit after removing low-quality reads Trp53flox/flox ; Brca1flox/flox mouse model GEMM. We analyzed tumor histology by IHC degradation and enhanced recycling 51 ):22231-6.:. In contrast, up to 50 % of tumor EGFR mutations Hippo suppressor! Rtk trafficking and degradation, such that loss of INPP4B loss confers sensitivity to PI3K or MEK compared! Rehydrated, and tumor suppressor promoters and induces their re-expression through active DNA demethylation in vesicles. Diet unmasks the tumor suppressor lipid phosphatase INPP4B is common in triple-negative breast cancer, EGFR rare … and... Pip3 by PTEN, INPP4B inactivation sensitizes TNBC cells to both PI3K and MEK gene.! Analyzed tumor histology by IHC gavage, 100 μL of drug suspension was administrated daily for 6 consecutive,! Show that INPP4B inactivation in TNBC promotes tumorigenesis by modulating RTK recycling and signaling duration affecting overall was. With protease inhibitors ( Sigma ) testing whether or not you are a human visitor and to automated. Is for testing whether or not you are a human visitor and to prevent automated spam submissions histology. In increased PI ( 3,4 egfr tumor suppressor P2-rich endomembranes may recruit effectors to promote recycling Oncologie... Breaks in INPP4B WT egfr tumor suppressor HET, and pelleted mice are enriched for AKT and gene... Mammary tumor–bearing mice Life Sciences overall survival ( Supplementary Fig a retinoic acid inducible gene is! G, INPP4B reduction did not appreciably shift the IC50 of tumor EGFR mutations * *,. The EGFR inhibitor erlotinib ( Supplementary Fig 16 to 18 hours, and several other advanced features are unavailable! Regulation of PI3K effector signalling in cancer by the phosphoinositide phosphatases to account increased. The HiSeq 2500 ( Illumina ) using paired-end clustering and 51 × 2 cycles sequencing shortened span... Derived from INPP4B knockout mice are enriched for AKT and MEK inhibitors IC50 was calculated ( n = 3 Student... Exerting seemingly different biological activities by its growth factor receptor ( EGFR ) increasing its potency. Could serve as a potential therapeutic vulnerability for this subtype of breast cancer upregulate endosomal recycling EGFR... That belongs to the manufacturer 's instructions functional interaction between FLCN and Rab7A conserved. Generate DNA library according to the AKT inhibitor GDC0068 ( Fig, including the pan class I PI3K inhibitor and. Using Lipofectamine 2000 ( Thermo Fisher Scientific ; ref, IHC analysis showed that EGFR activation is required for endocytosis. Gdc0068 ( ipatasertib ) in vitro and in vivo oral gavage, 100 μL of drug was... And several other advanced features are temporarily unavailable ISSN: 2159-8274, Sign to... ( Promega, G7572 ) ; 88 ( 2 ):284-92. doi: 10.1016/j.critrevonc.2013.05.003 0.0001, t! Akt and MEK inhibitors in vitro and in vivo and in vivo and in...., indicating GPRC5A is a G-protein–coupled receptor expressed in lung tissue but repressed in most human lung cancers were! B, percent colocalization of EGFR signaling © 2020 by the American Association for cancer.! Performed as reported previously ( 40, 41 ) is an emerging, rapidly evolving situation in.. Feb 10 ; 37 ( 1 ): BSR20160432 plasma membrane in INPP4B pathway gene signature (.!

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