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HCT 116

RRID:CVCL_0291

Organism

Homo sapiens

Comments

Part of: AstraZeneca Colorectal cell line (AZCL) panel. Part of: Cancer Cell Line Encyclopedia (CCLE) project. Part of: COSMIC cell lines project. Part of: ENCODE project common cell types; tier 3. Part of: FGFR genetic alteration cell panel (ATCC TCP-1034). Part of: JFCR39 cancer cell line panel. Part of: KuDOS 95 cell line panel. Part of: MD Anderson Cell Lines Project. Part of: NCI-60 cancer cell line panel. Doubling time: 36 hours (PubMed=25984343); 17.1 hours (PubMed=22628656); 17.4 hours (NCI-DTP); ~25-48 hours (DSMZ); ~21 hours (PBCF). HLA typing: A*01,02; B*18,45:01; C*05,07; DPB1*03:01:01,04:02; DQB1*02,03 (PubMed=15748285). HLA typing: A*01:01,02:01; B*45:01,18:01; C*05:01,07:01; DQA1*05:02,05:02; DQB1*03:09,02:02; DRB1*03:05,11:02 (PubMed=25960936). HLA typing: A*01:01,02:01; B*45:01; C*05:01,07:01 (PubMed=9178645). Microsatellite instability: Instable (MSI-high) (PubMed=9000147; PubMed=10674020; PubMed=23671654; PubMed=24042735; PubMed=25926053; PubMed=28683746; Sanger). Sequence variation: Homozygous for ACVR2A p.Lys437fs*5 (c.1310delA) (PubMed=12615714). Sequence variation: Heterozygous for BRCA2 p.Ile2675fs*6 (c.8021_8022insA) (PubMed=17088437). Sequence variation: Heterozygous for CDKN2A p.Arg24fs*20 (c.68_69insG) and p.Glu74fs*15 (c.220delG) (PubMed=17088437). Sequence variation: Heterozygous for CTNNB1 p.Ser45del (c.133_135delTCT) (ATCC; PubMed=9294210; PubMed=24755471). Sequence variation: Hemizygous for EP300 c.6294delA (PubMed=10700188). Sequence variation: Heterozygous for KRAS p.Gly13Asp (c.38G>A) (PubMed=12068308; PubMed=17088437; PubMed=20570890; PubMed=24755471; PubMed=28683746). Sequence variation: Heterozygous for PIK3CA p.His1047Arg (c.3140A>G) (PubMed=17088437; PubMed=20570890; PubMed=24755471; PubMed=28683746). Sequence variation: Homozygous for TGFBR2 p.Lys128Serfs*35 (c.383delA) (PubMed=12615714). Sequence variation: Has no TP53 mutation (PubMed=15900046; PubMed=16418264). Omics: Array-based CGH. Omics: CNV analysis. Omics: Deep exome analysis. Omics: Deep phosphoproteome analysis. Omics: Deep proteome analysis. Omics: Deep quantitative phosphoproteome analysis. Omics: Deep quantitative proteome analysis. Omics: Deep RNAseq analysis. Omics: DNA methylation analysis. Omics: Fluorescence phenotype profiling. Omics: H3K4me3 ChIP-seq epigenome analysis. Omics: HLA class I peptidome analysis by proteomics. Omics: lncRNA expression profiling. Omics: Metabolome analysis. Omics: miRNA expression profiling. Omics: N-glycan profiling. Omics: Protein expression by reverse-phase protein arrays. Omics: shRNA library screening. Omics: SNP array analysis. Omics: Transcriptome analysis. Genome ancestry: African=0%; Native American=0.08%; East Asian, North=1.16%; East Asian, South=0%; South Asian=0.97%; European, North=64.85%; European, South=32.93% (PubMed=30894373). Misspelling: HCT-166; In BTO BTO:0004743, now obsoleted. Misspelling: HTC 116; Occasionally. Misspelling: HTC-116; Occasionally. Misspelling: Htc-116; Occasionally. Misspelling: HTC116; Occasionally. Misspelling: HTC1116; Occasionally. DT Created: 04-04-12; Last updated: 24-05-19; Version: 30

Proper Citation

RCB Cat# RCB2979, RRID:CVCL_0291

Category

Cancer cell line DT Created: 04-04-12; Last updated: 24-05-19; Version: 30

Sex

DT Created: 04-04-12; Last updated: 24-05-19; Version: 30

Synonyms

HCT-116, HCT.116, HCT_116, HCT116, CoCL2 DT Created: 04-04-12, Last updated: 24-05-19, Version: 30

Vendor

RCB

Cat Num

RCB2979

Cross References

BTO; BTO:0001109 CLO; CLO_0003665 EFO; EFO_0002824 MCCL; MCC:0000181 CLDB; cl1574 CLDB; cl1575 CLDB; cl5198 4DN; 4DNSRMYUIVGD AddexBio; C0009005/41 ArrayExpress; E-MTAB-2706 ArrayExpress; E-MTAB-2770 ArrayExpress; E-MTAB-3610 ATCC; CCL-247 BCRC; 60349 BCRJ; 0288 BioSample; SAMN03470980 BioSample; SAMN03471481 BioSample; SAMN03473487 BioSample; SAMN05292433 CCLE; HCT116_LARGE_INTESTINE CCRID; 3111C0001CCC000158 CCRID; 3111C0001CCC000331 CCRID; 3131C0001000700099 Cell_Model_Passport; SIDM00783 ChEMBL-Cells; CHEMBL3308372 ChEMBL-Targets; CHEMBL394 CLS; 300195/p19841_HCT116.html ColonAtlas; HCT116 Cosmic; 711257 Cosmic; 720329 Cosmic; 724840 Cosmic; 869794 Cosmic; 870449 Cosmic; 873701 Cosmic; 875296 Cosmic; 875425 Cosmic; 875847 Cosmic; 876644 Cosmic; 876706 Cosmic; 887223 Cosmic; 889535 Cosmic; 897457 Cosmic; 902790 Cosmic; 905936 Cosmic; 913887 Cosmic; 934564 Cosmic; 947355 Cosmic; 948126 Cosmic; 948861 Cosmic; 985996 Cosmic; 995410 Cosmic; 1043815 Cosmic; 1044257 Cosmic; 1045410 Cosmic; 1057752 Cosmic; 1066205 Cosmic; 1067220 Cosmic; 1092600 Cosmic; 1102383 Cosmic; 1122325 Cosmic; 1131683 Cosmic; 1132568 Cosmic; 1132689 Cosmic; 1154644 Cosmic; 1175840 Cosmic; 1176588 Cosmic; 1183769 Cosmic; 1184080 Cosmic; 1184333 Cosmic; 1187306 Cosmic; 1218874 Cosmic; 1223141 Cosmic; 1305353 Cosmic; 1310945 Cosmic; 1332008 Cosmic; 1436021 Cosmic; 1466804 Cosmic; 1466815 Cosmic; 1479594 Cosmic; 1486132 Cosmic; 1519359 Cosmic; 1524003 Cosmic; 1524333 Cosmic; 1552179 Cosmic; 1571772 Cosmic; 1609495 Cosmic; 1672408 Cosmic; 1676727 Cosmic; 1708402 Cosmic; 1805253 Cosmic; 1927246 Cosmic; 1945865 Cosmic; 1995437 Cosmic; 1998442 Cosmic; 2036656 Cosmic; 2046556 Cosmic; 2052593 Cosmic; 2145576 Cosmic; 2301978 Cosmic; 2389569 Cosmic; 2433755 Cosmic; 2464667 Cosmic; 2550353 Cosmic; 2646766 Cosmic; 2651864 Cosmic; 2667881 Cosmic; 2667972 Cosmic; 2668248 Cosmic; 2727479 Cosmic-CLP; 905936 DSMZ; ACC-581 ECACC; 91091005 ENCODE; ENCBS004UZY ENCODE; ENCBS009DQE ENCODE; ENCBS257IHV ENCODE; ENCBS378ENC ENCODE; ENCBS389ENC ENCODE; ENCBS409ENC ENCODE; ENCBS409MTT ENCODE; ENCBS431ENC ENCODE; ENCBS471AAA ENCODE; ENCBS472AAA ENCODE; ENCBS475AAA ENCODE; ENCBS476AAA ENCODE; ENCBS494OPB ENCODE; ENCBS650OZU ENCODE; ENCBS847SOB GDSC; 905936 GEO; GSM810 GEO; GSM2143 GEO; GSM50188 GEO; GSM50252 GEO; GSM115118 GEO; GSM206501 GEO; GSM274713 GEO; GSM274714 GEO; GSM274726 GEO; GSM481405 GEO; GSM513818 GEO; GSM514292 GEO; GSM580039 GEO; GSM580040 GEO; GSM741266 GEO; GSM750773 GEO; GSM750793 GEO; GSM784012 GEO; GSM799331 GEO; GSM799394 GEO; GSM827446 GEO; GSM846355 GEO; GSM887062 GEO; GSM888132 GEO; GSM945287 GEO; GSM945304 GEO; GSM1153399 GEO; GSM1181250 GEO; GSM1181367 GEO; GSM1346874 GEO; GSM1374516 GEO; GSM1374517 GEO; GSM1374518 GEO; GSM1404391 GEO; GSM1404392 GEO; GSM1448092 GEO; GSM1669868 GEO; GSM2124655 GEO; GSM2550001 IARC_TP53; 21064 ICLC; HTL95025 IZSLER; BS TCL 100 KCB; KCB 200706YJ KCLB; 10247 LINCS_LDP; LCL-1161 Lonza; 691 MeSH; D045325 MetaboLights; MTBLS227 NCBI_Iran; C570 NCI-DTP; HCT-116 PRIDE; PXD000089 PRIDE; PXD000394 PRIDE; PXD001352 PRIDE; PXD001426 PRIDE; PXD001546 PRIDE; PXD001550 PRIDE; PXD002793 PRIDE; PXD005235 PRIDE; PXD005354 PRIDE; PXD005942 RCB; RCB2979 SKY/M-FISH/CGH; 1687 SKY/M-FISH/CGH; 2778 SKY/M-FISH/CGH; 2785 SKY/M-FISH/CGH; 3883 TOKU-E; 1422 Wikidata; Q28334584 DT Created: 04-04-12; Last updated: 24-05-19; Version: 30

Dependence on the Pyrimidine Biosynthetic Enzyme DHODH Is a Synthetic Lethal Vulnerability in Mutant KRAS-Driven Cancers.

  • Koundinya M
  • Cell Chem Biol
  • 2018 Jun 21

Literature context:


Abstract:

Activating KRAS mutations are major oncogenic drivers in multiple tumor types. Synthetic lethal screens have previously been used to identify targets critical for the survival of KRAS mutant cells, but their application to drug discovery has proven challenging, possibly due in part to a failure of monolayer cultures to model tumor biology. Here, we report the results of a high-throughput synthetic lethal screen for small molecules that selectively inhibit the growth of KRAS mutant cell lines in soft agar. Chemoproteomic profiling identifies the target of the most KRAS-selective chemical series as dihydroorotate dehydrogenase (DHODH). DHODH inhibition is shown to perturb multiple metabolic pathways. In vivo preclinical studies demonstrate strong antitumor activity upon DHODH inhibition in a pancreatic tumor xenograft model.

Funding information:
  • Medical Research Council - (United Kingdom)

A Designed Peptide Targets Two Types of Modifications of p53 with Anti-cancer Activity.

  • Liang L
  • Cell Chem Biol
  • 2018 Jun 21

Literature context:


Abstract:

Many cancer-related proteins are controlled by composite post-translational modifications (PTMs), but prevalent strategies only target one type of modification. Here we describe a designed peptide that controls two types of modifications of the p53 tumor suppressor, based on the discovery of a protein complex that suppresses p53 (suppresome). We found that Morn3, a cancer-testis antigen, recruits different PTM enzymes, such as sirtuin deacetylase and ubiquitin ligase, to confer composite modifications on p53. The molecular functions of Morn3 were validated through in vivo assays and chemico-biological intervention. A rationally designed Morn3-targeting peptide (Morncide) successfully activated p53 and suppressed tumor growth. These findings shed light on the regulation of protein PTMs and present a strategy for targeting two modifications with one molecule.

Funding information:
  • NCI NIH HHS - K08 CA102545(United States)

A Rapid and Precise Mutation-Activated Fluorescence Reporter for Analyzing Acute Mutagenesis Frequency.

  • Birnbaum MD
  • Cell Chem Biol
  • 2018 May 28

Literature context:


Abstract:

Mutagenesis reporters are critical for quantifying genome stability. However, current methods rely on cell survival/death to report mutation, which takes weeks and prevents evaluation of acute or time-dependent changes. Existing methods also have other limitations, such as cell type restrictions. Using our discovery that mCherryFP fluorescence depends on residue Trp98, we replaced this codon with a stop codon to generate a mutation biosensor (termed CherryOFF), with a green fluorescence protein (GFP) as an internal control. We found that the red fluorescence of this biosensor is activated by a specific A/T-G/C nucleotide transition. Compared with the established hypoxanthine phosphoribosyl transferase assay, our reporter has similar or better ability to detect changes of mutation frequency induced by physical/chemical mutagens or manipulation of mutation-related genes. Furthermore, CherryOFF-GFP can report mutagenesis independently of cell-death events, can be adapted to many cell types, and can generate readouts within 1 day for the measurement of acute or time-dependent events.

Funding information:
  • NIDDK NIH HHS - DK056246(United States)

KREMEN1 Is a Host Entry Receptor for a Major Group of Enteroviruses.

  • Staring J
  • Cell Host Microbe
  • 2018 May 9

Literature context:


Abstract:

Human type A Enteroviruses (EV-As) cause diseases ranging from hand-foot-and-mouth disease to poliomyelitis-like disease. Although cellular receptors are identified for some EV-As, they remain elusive for the majority of EV-As. We identify the cell surface molecule KREMEN1 as an entry receptor for coxsackievirus A10 (CV-A10). Whereas loss of KREMEN1 renders cells resistant to CV-A10 infection, KREMEN1 overexpression enhances CV-A10 binding to the cell surface and increases susceptibility to infection, indicating that KREMEN1 is a rate-limiting factor for CV-A10 infection. Furthermore, the extracellular domain of KREMEN1 binds CV-A10 and functions as a neutralizing agent during infection. Kremen-deficient mice are resistant to CV-A10-induced lethal paralysis, emphasizing the relevance of Kremen for infection in vivo. KREMEN1 is also essential for infection by a phylogenetic and pathogenic related group of EV-As. Collectively these findings highlight the importance of KREMEN1 for these emerging pathogens and its potential as an antiviral therapeutic target.

Funding information:
  • NIGMS NIH HHS - GM083863(United States)

GPR68 Senses Flow and Is Essential for Vascular Physiology.

  • Xu J
  • Cell
  • 2018 Apr 19

Literature context:


Abstract:

Mechanotransduction plays a crucial role in vascular biology. One example of this is the local regulation of vascular resistance via flow-mediated dilation (FMD). Impairment of this process is a hallmark of endothelial dysfunction and a precursor to a wide array of vascular diseases, such as hypertension and atherosclerosis. Yet the molecules responsible for sensing flow (shear stress) within endothelial cells remain largely unknown. We designed a 384-well screening system that applies shear stress on cultured cells. We identified a mechanosensitive cell line that exhibits shear stress-activated calcium transients, screened a focused RNAi library, and identified GPR68 as necessary and sufficient for shear stress responses. GPR68 is expressed in endothelial cells of small-diameter (resistance) arteries. Importantly, Gpr68-deficient mice display markedly impaired acute FMD and chronic flow-mediated outward remodeling in mesenteric arterioles. Therefore, GPR68 is an essential flow sensor in arteriolar endothelium and is a critical signaling component in cardiovascular pathophysiology.

Funding information:
  • NCI NIH HHS - U24 CA126543(United States)

Systematic Characterization of Stress-Induced RNA Granulation.

  • Namkoong S
  • Mol. Cell
  • 2018 Apr 5

Literature context:


Abstract:

Upon stress, cytoplasmic mRNA is sequestered to insoluble ribonucleoprotein (RNP) granules, such as the stress granule (SG). Partially due to the belief that translationally suppressed mRNAs are recruited to SGs in bulk, stress-induced dynamic redistribution of mRNA has not been thoroughly characterized. Here, we report that endoplasmic reticulum (ER) stress targets only a small subset of translationally suppressed mRNAs into the insoluble RNP granule fraction (RG). This subset, characterized by extended length and adenylate-uridylate (AU)-rich motifs, is highly enriched with genes critical for cell survival and proliferation. This pattern of RG targeting was conserved for two other stress types, heat shock and arsenite toxicity, which induce distinct responses in the total cytoplasmic transcriptome. Nevertheless, stress-specific RG-targeting motifs, such as guanylate-cytidylate (GC)-rich motifs in heat shock, were also identified. Previously underappreciated, transcriptome profiling in the RG may contribute to understanding human diseases associated with RNP dysfunction, such as cancer and neurodegeneration.

Funding information:
  • NIAID NIH HHS - R01 AI043477(United States)

PAF-Myc-Controlled Cell Stemness Is Required for Intestinal Regeneration and Tumorigenesis.

  • Kim MJ
  • Dev. Cell
  • 2018 Mar 12

Literature context:


Abstract:

The underlying mechanisms of how self-renewing cells are controlled in regenerating tissues and cancer remain ambiguous. PCNA-associated factor (PAF) modulates DNA repair via PCNA. Also, PAF hyperactivates Wnt/β-catenin signaling independently of PCNA interaction. We found that PAF is expressed in intestinal stem and progenitor cells (ISCs and IPCs) and markedly upregulated during intestinal regeneration and tumorigenesis. Whereas PAF is dispensable for intestinal homeostasis, upon radiation injury, genetic ablation of PAF impairs intestinal regeneration along with the severe loss of ISCs and Myc expression. Mechanistically, PAF conditionally occupies and transactivates the c-Myc promoter, which induces the expansion of ISCs/IPCs during intestinal regeneration. In mouse models, PAF knockout inhibits Apc inactivation-driven intestinal tumorigenesis with reduced tumor cell stemness and suppressed Wnt/β-catenin signaling activity, supported by transcriptome profiling. Collectively, our results unveil that the PAF-Myc signaling axis is indispensable for intestinal regeneration and tumorigenesis by positively regulating self-renewing cells.

Funding information:
  • NCI NIH HHS - 2P01CA098912(United States)
  • NCI NIH HHS - P30 CA016672()
  • NCI NIH HHS - P50 CA098258()
  • NCI NIH HHS - R01 CA193297()

Transcriptional Regulation of the Warburg Effect in Cancer by SIX1.

  • Li L
  • Cancer Cell
  • 2018 Mar 12

Literature context:


Abstract:

Aerobic glycolysis (the Warburg effect) facilitates tumor growth, and drugs targeting aerobic glycolysis are being developed. However, how the Warburg effect is directly regulated is largely unknown. Here we show that transcription factor SIX1 directly increases the expression of many glycolytic genes, promoting the Warburg effect and tumor growth in vitro and in vivo. SIX1 regulates glycolysis through HBO1 and AIB1 histone acetyltransferases. Cancer-related SIX1 mutation increases its ability to promote aerobic glycolysis and tumor growth. SIX1 glycolytic function is directly repressed by microRNA-548a-3p, which is downregulated, inversely correlates with SIX1, and is a good predictor of prognosis in breast cancer patients. Thus, the microRNA-548a-3p/SIX1 axis strongly links aerobic glycolysis to carcinogenesis and may become a promising cancer therapeutic target.

Funding information:
  • NHLBI NIH HHS - HL084312(United States)

Explosive mutation accumulation triggered by heterozygous human Pol ε proofreading-deficiency is driven by suppression of mismatch repair.

  • Hodel KP
  • Elife
  • 2018 Feb 28

Literature context:


Abstract:

Tumors defective for DNA polymerase (Pol) ε proofreading have the highest tumor mutation burden identified. A major unanswered question is whether loss of Pol ε proofreading by itself is sufficient to drive this mutagenesis, or whether additional factors are necessary. To address this, we used a combination of next generation sequencing and in vitro biochemistry on human cell lines engineered to have defects in Pol ε proofreading and mismatch repair. Absent mismatch repair, monoallelic Pol ε proofreading deficiency caused a rapid increase in a unique mutation signature, similar to that observed in tumors from patients with biallelic mismatch repair deficiency and heterozygous Pol ε mutations. Restoring mismatch repair was sufficient to suppress the explosive mutation accumulation. These results strongly suggest that concomitant suppression of mismatch repair, a hallmark of colorectal and other aggressive cancers, is a critical force for driving the explosive mutagenesis seen in tumors expressing exonuclease-deficient Pol ε.

Funding information:
  • National Institute of Environmental Health Sciences - NIH P20 RR020152()
  • National Institute of Environmental Health Sciences - NIH R00 ES016780()
  • National Institute of Environmental Health Sciences - NIH R01ES028271()
  • National Institute of Environmental Health Sciences - NIH R56ES026821()
  • NCI NIH HHS - R01 CA129925()
  • NCRR NIH HHS - P20 RR020152()
  • NIAID NIH HHS - R56 AI028847(United States)
  • NIEHS NIH HHS - R00 ES016780()
  • NIEHS NIH HHS - R01 ES028271()
  • NIEHS NIH HHS - R56 ES026821()
  • Tulane University - Stem Cell and Regenerative Medicine Faculty Grant()

Small interfering RNAs based on huntingtin trinucleotide repeats are highly toxic to cancer cells.

  • Murmann AE
  • EMBO Rep.
  • 2018 Feb 15

Literature context:


Abstract:

Trinucleotide repeat (TNR) expansions in the genome cause a number of degenerative diseases. A prominent TNR expansion involves the triplet CAG in the huntingtin (HTT) gene responsible for Huntington's disease (HD). Pathology is caused by protein and RNA generated from the TNR regions including small siRNA-sized repeat fragments. An inverse correlation between the length of the repeats in HTT and cancer incidence has been reported for HD patients. We now show that siRNAs based on the CAG TNR are toxic to cancer cells by targeting genes that contain long reverse complementary TNRs in their open reading frames. Of the 60 siRNAs based on the different TNRs, the six members in the CAG/CUG family of related TNRs are the most toxic to both human and mouse cancer cells. siCAG/CUG TNR-based siRNAs induce cell death in vitro in all tested cancer cell lines and slow down tumor growth in a preclinical mouse model of ovarian cancer with no signs of toxicity to the mice. We propose to explore TNR-based siRNAs as a novel form of anticancer reagents.

Funding information:
  • NCI NIH HHS - R01 CA167041()
  • NCI NIH HHS - R01CA127645(United States)
  • NCI NIH HHS - R35 CA197450()
  • NCI NIH HHS - T32 CA009560()
  • NCI NIH HHS - T32 CA070085()

mTORC1 Inactivation Promotes Colitis-Induced Colorectal Cancer but Protects from APC Loss-Dependent Tumorigenesis.

  • Brandt M
  • Cell Metab.
  • 2018 Jan 9

Literature context:


Abstract:

Dietary habits that can induce inflammatory bowel disease (IBD) are major colorectal cancer (CRC) risk factors, but mechanisms linking nutrients, IBD, and CRC are unknown. Using human data and mouse models, we show that mTORC1 inactivation-induced chromosomal instability impairs intestinal crypt proliferation and regeneration, CDK4/6 dependently. This triggers interleukin (IL)-6-associated reparative inflammation, inducing crypt hyper-proliferation, wound healing, and CRC. Blocking IL-6 signaling or reactivating mTORC1 reduces inflammation-induced CRC, so mTORC1 activation suppresses tumorigenesis in IBD. Conversely, mTORC1 inactivation is beneficial in APC loss-dependent CRC. Thus, IL-6 blockers or protein-rich-diet-linked mTORC1 activation may prevent IBD-associated CRC. However, abolishing mTORC1 can mitigate CRC in predisposed patients with APC mutations. Our work reveals mTORC1 oncogenic and tumor-suppressive roles in intestinal epithelium and avenues to optimized and personalized therapeutic regimens for CRC.

Funding information:
  • NIGMS NIH HHS - T32 GM007067(United States)

p53 orchestrates DNA replication restart homeostasis by suppressing mutagenic RAD52 and POLθ pathways.

  • Roy S
  • Elife
  • 2018 Jan 15

Literature context:


Abstract:

Classically, p53 tumor suppressor acts in transcription, apoptosis, and cell cycle arrest. Yet, replication-mediated genomic instability is integral to oncogenesis, and p53 mutations promote tumor progression and drug-resistance. By delineating human and murine separation-of-function p53 alleles, we find that p53 null and gain-of-function (GOF) mutations exhibit defects in restart of stalled or damaged DNA replication forks that drive genomic instability, which isgenetically separable from transcription activation. By assaying protein-DNA fork interactions in single cells, we unveil a p53-MLL3-enabled recruitment of MRE11 DNA replication restart nuclease. Importantly, p53 defects or depletion unexpectedly allow mutagenic RAD52 and POLθ pathways to hijack stalled forks, which we find reflected in p53 defective breast-cancer patient COSMIC mutational signatures. These data uncover p53 as a keystone regulator of replication homeostasis within a DNA restart network. Mechanistically, this has important implications for development of resistance in cancer therapy. Combined, these results define an unexpected role for p53-mediated suppression of replication genome instability.

Funding information:
  • NIAID NIH HHS - R01 AI038382(United States)

Targeting KRAS Mutant Cancers with a Covalent G12C-Specific Inhibitor.

  • Janes MR
  • Cell
  • 2018 Jan 25

Literature context:


Abstract:

KRASG12C was recently identified to be potentially druggable by allele-specific covalent targeting of Cys-12 in vicinity to an inducible allosteric switch II pocket (S-IIP). Success of this approach requires active cycling of KRASG12C between its active-GTP and inactive-GDP conformations as accessibility of the S-IIP is restricted only to the GDP-bound state. This strategy proved feasible for inhibiting mutant KRAS in vitro; however, it is uncertain whether this approach would translate to in vivo. Here, we describe structure-based design and identification of ARS-1620, a covalent compound with high potency and selectivity for KRASG12C. ARS-1620 achieves rapid and sustained in vivo target occupancy to induce tumor regression. We use ARS-1620 to dissect oncogenic KRAS dependency and demonstrate that monolayer culture formats significantly underestimate KRAS dependency in vivo. This study provides in vivo evidence that mutant KRAS can be selectively targeted and reveals ARS-1620 as representing a new generation of KRASG12C-specific inhibitors with promising therapeutic potential.

Funding information:
  • NCI NIH HHS - R01-CA093678(United States)

Endosomal Rab cycles regulate Parkin-mediated mitophagy.

  • Yamano K
  • Elife
  • 2018 Jan 23

Literature context:


Abstract:

Damaged mitochondria are selectively eliminated by mitophagy. Parkin and PINK1, gene products mutated in familial Parkinson's disease, play essential roles in mitophagy through ubiquitination of mitochondria. Cargo ubiquitination by E3 ubiquitin ligase Parkin is important to trigger selective autophagy. Although autophagy receptors recruit LC3-labeled autophagic membranes onto damaged mitochondria, how other essential autophagy units such as ATG9A-integrated vesicles are recruited remains unclear. Here, using mammalian cultured cells, we demonstrate that RABGEF1, the upstream factor of the endosomal Rab GTPase cascade, is recruited to damaged mitochondria via ubiquitin binding downstream of Parkin. RABGEF1 directs the downstream Rab proteins, RAB5 and RAB7A, to damaged mitochondria, whose associations are further regulated by mitochondrial Rab-GAPs. Furthermore, depletion of RAB7A inhibited ATG9A vesicle assembly and subsequent encapsulation of the mitochondria by autophagic membranes. These results strongly suggest that endosomal Rab cycles on damaged mitochondria are a crucial regulator of mitophagy through assembling ATG9A vesicles.

Funding information:
  • Japan Science and Technology Agency - JPMJCR13M7(International)
  • Japan Society for the Promotion of Science - 16K15095(International)
  • Japan Society for the Promotion of Science - JP15H01196(International)
  • Japan Society for the Promotion of Science - JP16K18545(International)
  • Japan Society for the Promotion of Science - JP26000014(International)
  • Japan Society for the Promotion of Science - JP26111729(International)
  • Japan Society for the Promotion of Science - JP26840033(International)
  • NIDCR NIH HHS - R03 DE018415-02(United States)
  • NINDS NIH HHS - Intramural program(United States)

SIRT2 and lysine fatty acylation regulate the transforming activity of K-Ras4a.

  • Jing H
  • Elife
  • 2017 Dec 14

Literature context:


Abstract:

Ras proteins play vital roles in numerous biological processes and Ras mutations are found in many human tumors. Understanding how Ras proteins are regulated is important for elucidating cell signaling pathways and identifying new targets for treating human diseases. Here we report that one of the K-Ras splice variants, K-Ras4a, is subject to lysine fatty acylation, a previously under-studied protein post-translational modification. Sirtuin 2 (SIRT2), one of the mammalian nicotinamide adenine dinucleotide (NAD)-dependent lysine deacylases, catalyzes the removal of fatty acylation from K-Ras4a. We further demonstrate that SIRT2-mediated lysine defatty-acylation promotes endomembrane localization of K-Ras4a, enhances its interaction with A-Raf, and thus promotes cellular transformation. Our study identifies lysine fatty acylation as a previously unknown regulatory mechanism for the Ras family of GTPases that is distinct from cysteine fatty acylation. These findings highlight the biological significance of lysine fatty acylation and sirtuin-catalyzed protein lysine defatty-acylation.

Funding information:
  • NCRR NIH HHS - S10 RR025502()
  • NIAID NIH HHS - R03 AI078348(United States)
  • NIGMS NIH HHS - R01 GM121540()
  • NIH HHS - S10 OD017992()

USP7-Specific Inhibitors Target and Modify the Enzyme's Active Site via Distinct Chemical Mechanisms.

  • Pozhidaeva A
  • Cell Chem Biol
  • 2017 Dec 21

Literature context:


Abstract:

USP7 is a deubiquitinating enzyme that plays a pivotal role in multiple oncogenic pathways and therefore is a desirable target for new anti-cancer therapies. However, the lack of structural information about the USP7-inhibitor interactions has been a critical gap in the development of potent inhibitors. USP7 is unique among USPs in that its active site is catalytically incompetent, and is postulated to rearrange into a productive conformation only upon binding to ubiquitin. Surprisingly, we found that ubiquitin alone does not induce an active conformation in solution. Using a combination of nuclear magnetic resonance, mass spectrometry, computational modeling, and cell-based assays, we found that DUB inhibitors P22077 and P50429 covalently modify the catalytic cysteine of USP7 and induce a conformational switch in the enzyme associated with active site rearrangement. This work represents the first experimental insights into USP7 activation and inhibition and provides a structural basis for rational development of potent anti-cancer therapeutics.

Funding information:
  • NHLBI NIH HHS - F30 HL095280(United States)

A Method for the Acute and Rapid Degradation of Endogenous Proteins.

  • Clift D
  • Cell
  • 2017 Dec 14

Literature context:


Abstract:

Methods for the targeted disruption of protein function have revolutionized science and greatly expedited the systematic characterization of genes. Two main approaches are currently used to disrupt protein function: DNA knockout and RNA interference, which act at the genome and mRNA level, respectively. A method that directly alters endogenous protein levels is currently not available. Here, we present Trim-Away, a technique to degrade endogenous proteins acutely in mammalian cells without prior modification of the genome or mRNA. Trim-Away harnesses the cellular protein degradation machinery to remove unmodified native proteins within minutes of application. This rapidity minimizes the risk that phenotypes are compensated and that secondary, non-specific defects accumulate over time. Because Trim-Away utilizes antibodies, it can be applied to a wide range of target proteins using off-the-shelf reagents. Trim-Away allows the study of protein function in diverse cell types, including non-dividing primary cells where genome- and RNA-targeting methods are limited.

Funding information:
  • NIDCD NIH HHS - P30 DC04657(United States)

iASPP Is an Antioxidative Factor and Drives Cancer Growth and Drug Resistance by Competing with Nrf2 for Keap1 Binding.

  • Ge W
  • Cancer Cell
  • 2017 Nov 13

Literature context:


Abstract:

Reactive oxygen species (ROS) have emerged as important signaling molecules that play crucial roles in carcinogenesis and cytotoxic responses. Nrf2 is the master regulator of ROS balance. Thus, uncovering mechanisms of Nrf2 regulation is important for the development of alternative treatment strategies for cancers. Here, we demonstrate that iASPP, a known p53 inhibitor, lowers ROS independently of p53. Mechanistically, iASPP competes with Nrf2 for Keap1 binding via a DLT motif, leading to decreased Nrf2 ubiquitination and increased Nrf2 accumulation, nuclear translocation, and antioxidative transactivation. This iASPP-Keap1-Nrf2 axis promotes cancer growth and drug resistance both in vitro and in vivo. Thus, iASPP is an antioxidative factor and represents a promising target to improve cancer treatment, regardless of p53 status.

Funding information:
  • NCI NIH HHS - R01CA169200(United States)

Widespread Post-transcriptional Attenuation of Genomic Copy-Number Variation in Cancer.

  • Gonçalves E
  • Cell Syst
  • 2017 Oct 25

Literature context:


Abstract:

Copy-number variations (CNVs) are ubiquitous in cancer and often act as driver events, but the effects of CNVs on the proteome of tumors are poorly understood. Here, we analyze recently published genomics, transcriptomics, and proteomics datasets made available by CPTAC and TCGA consortia on 282 breast, ovarian, and colorectal tumor samples to investigate the impact of CNVs in the proteomes of these cells. We found that CNVs are buffered by post-transcriptional regulation in 23%-33% of proteins that are significantly enriched in protein complex members. Our analyses show that complex subunits are highly co-regulated, and some act as rate-limiting steps of complex assembly, as their depletion induces decreased abundance of other complex members. We identified 48 such rate-limiting interactions and experimentally confirmed our predictions on the interactions of AP3B1 with AP3M1 and GTF2E2 with GTF2E1. This study highlights the importance of post-transcriptional mechanisms in cancer that allow cells to cope with their altered genomes.

Coordinated Splicing of Regulatory Detained Introns within Oncogenic Transcripts Creates an Exploitable Vulnerability in Malignant Glioma.

  • Braun CJ
  • Cancer Cell
  • 2017 Oct 9

Literature context:


Abstract:

Glioblastoma (GBM) is a devastating malignancy with few therapeutic options. We identify PRMT5 in an in vivo GBM shRNA screen and show that PRMT5 knockdown or inhibition potently suppresses in vivo GBM tumors, including patient-derived xenografts. Pathway analysis implicates splicing in cellular PRMT5 dependency, and we identify a biomarker that predicts sensitivity to PRMT5 inhibition. We find that PRMT5 deficiency primarily disrupts the removal of detained introns (DIs). This impaired DI splicing affects proliferation genes, whose downregulation coincides with cell cycle defects, senescence and/or apoptosis. We further show that DI programs are evolutionarily conserved and operate during neurogenesis, suggesting that they represent a physiological regulatory mechanism. Collectively, these findings reveal a PRMT5-regulated DI-splicing program as an exploitable cancer vulnerability.

A Predictive Model for Selective Targeting of the Warburg Effect through GAPDH Inhibition with a Natural Product.

  • Liberti MV
  • Cell Metab.
  • 2017 Oct 3

Literature context:


Abstract:

Targeted cancer therapies that use genetics are successful, but principles for selectively targeting tumor metabolism that is also dependent on the environment remain unknown. We now show that differences in rate-controlling enzymes during the Warburg effect (WE), the most prominent hallmark of cancer cell metabolism, can be used to predict a response to targeting glucose metabolism. We establish a natural product, koningic acid (KA), to be a selective inhibitor of GAPDH, an enzyme we characterize to have differential control properties over metabolism during the WE. With machine learning and integrated pharmacogenomics and metabolomics, we demonstrate that KA efficacy is not determined by the status of individual genes, but by the quantitative extent of the WE, leading to a therapeutic window in vivo. Thus, the basis of targeting the WE can be encoded by molecular principles that extend beyond the status of individual genes.

Funding information:
  • NCI NIH HHS - R00 CA168997()
  • NCI NIH HHS - R01 CA174643()
  • NCI NIH HHS - R01 CA193256()
  • NIDDK NIH HHS - R01 DK105550()
  • NIGMS NIH HHS - T32 GM007273()
  • NIGMS NIH HHS - T32 GM008500()

A Peptide Encoded by a Putative lncRNA HOXB-AS3 Suppresses Colon Cancer Growth.

  • Huang JZ
  • Mol. Cell
  • 2017 Oct 5

Literature context:


Abstract:

A substantial fraction of eukaryotic transcripts are considered long non-coding RNAs (lncRNAs), which regulate various hallmarks of cancer. Here, we discovered that the lncRNA HOXB-AS3 encodes a conserved 53-aa peptide. The HOXB-AS3 peptide, not lncRNA, suppresses colon cancer (CRC) growth. Mechanistically, the HOXB-AS3 peptide competitively binds to the ariginine residues in RGG motif of hnRNP A1 and antagonizes the hnRNP A1-mediated regulation of pyruvate kinase M (PKM) splicing by blocking the binding of the ariginine residues in RGG motif of hnRNP A1 to the sequences flanking PKM exon 9, ensuring the formation of lower PKM2 and suppressing glucose metabolism reprogramming. CRC patients with low levels of HOXB-AS3 peptide have poorer prognoses. Our study indicates that the loss of HOXB-AS3 peptide is a critical oncogenic event in CRC metabolic reprogramming. Our findings uncover a complex regulatory mechanism of cancer metabolism reprogramming orchestrated by a peptide encoded by an lncRNA.

Many si/shRNAs can kill cancer cells by targeting multiple survival genes through an off-target mechanism.

  • Putzbach W
  • Elife
  • 2017 Oct 24

Literature context:


Abstract:

Over 80% of multiple-tested siRNAs and shRNAs targeting CD95 or CD95 ligand (CD95L) induce a form of cell death characterized by simultaneous activation of multiple cell death pathways preferentially killing transformed and cancer stem cells. We now show these si/shRNAs kill cancer cells through canonical RNAi by targeting the 3'UTR of critical survival genes in a unique form of off-target effect we call DISE (death induced by survival gene elimination). Drosha and Dicer-deficient cells, devoid of most miRNAs, are hypersensitive to DISE, suggesting cellular miRNAs protect cells from this form of cell death. By testing 4666 shRNAs derived from the CD95 and CD95L mRNA sequences and an unrelated control gene, Venus, we have identified many toxic sequences - most of them located in the open reading frame of CD95L. We propose that specific toxic RNAi-active sequences present in the genome can kill cancer cells.

Funding information:
  • NCI NIH HHS - R35 CA197450()
  • NCI NIH HHS - R50 CA211271()
  • NCI NIH HHS - T32 CA009560()
  • NCI NIH HHS - T32 CA070085()

Tumour-derived PGD2 and NKp30-B7H6 engagement drives an immunosuppressive ILC2-MDSC axis.

  • Trabanelli S
  • Nat Commun
  • 2017 Sep 19

Literature context:


Abstract:

Group 2 innate lymphoid cells (ILC2s) are involved in human diseases, such as allergy, atopic dermatitis and nasal polyposis, but their function in human cancer remains unclear. Here we show that, in acute promyelocytic leukaemia (APL), ILC2s are increased and hyper-activated through the interaction of CRTH2 and NKp30 with elevated tumour-derived PGD2 and B7H6, respectively. ILC2s, in turn, activate monocytic myeloid-derived suppressor cells (M-MDSCs) via IL-13 secretion. Upon treating APL with all-trans retinoic acid and achieving complete remission, the levels of PGD2, NKp30, ILC2s, IL-13 and M-MDSCs are restored. Similarly, disruption of this tumour immunosuppressive axis by specifically blocking PGD2, IL-13 and NKp30 partially restores ILC2 and M-MDSC levels and results in increased survival. Thus, using APL as a model, we uncover a tolerogenic pathway that may represent a relevant immunosuppressive, therapeutic targetable, mechanism operating in various human tumour types, as supported by our observations in prostate cancer.Group 2 innate lymphoid cells (ILC2s) modulate inflammatory and allergic responses, but their function in cancer immunity is still unclear. Here the authors show that, in acute promyelocytic leukaemia, tumour-activated ILC2s secrete IL-13 to induce myeloid-derived suppressor cells and support tumour growth.

The DREAM complex through its subunit Lin37 cooperates with Rb to initiate quiescence.

  • Mages CF
  • Elife
  • 2017 Sep 18

Literature context:


Abstract:

The retinoblastoma Rb protein is an important factor controlling the cell cycle. Yet, mammalian cells carrying Rb deletions are still able to arrest under growth-limiting conditions. The Rb-related proteins p107 and p130, which are components of the DREAM complex, had been suggested to be responsible for a continued ability to arrest by inhibiting E2f activity and by recruiting chromatin-modifying enzymes. Here, we show that p130 and p107 are not sufficient for DREAM-dependent repression. We identify the MuvB protein Lin37 as an essential factor for DREAM function. Cells not expressing Lin37 proliferate normally, but DREAM completely loses its ability to repress genes in G0/G1 while all remaining subunits, including p130/p107, still bind to target gene promoters. Furthermore, cells lacking both Rb and Lin37 are incapable of exiting the cell cycle. Thus, Lin37 is an essential component of DREAM that cooperates with Rb to induce quiescence.

Potent and Selective Covalent Quinazoline Inhibitors of KRAS G12C.

  • Zeng M
  • Cell Chem Biol
  • 2017 Aug 17

Literature context:


Abstract:

Targeted covalent small molecules have shown promise for cancers driven by KRAS G12C. Allosteric compounds that access an inducible pocket formed by movement of a dynamic structural element in KRAS, switch II, have been reported, but these compounds require further optimization to enable their advancement into clinical development. We demonstrate that covalent quinazoline-based switch II pocket (SIIP) compounds effectively suppress GTP loading of KRAS G12C, MAPK phosphorylation, and the growth of cancer cells harboring G12C. Notably we find that adding an amide substituent to the quinazoline scaffold allows additional interactions with KRAS G12C, and remarkably increases the labeling efficiency, potency, and selectivity of KRAS G12C inhibitors. Structural studies using X-ray crystallography reveal a new conformation of SIIP and key interactions made by substituents located at the quinazoline 2-, 4-, and 7-positions. Optimized lead compounds in the quinazoline series selectively inhibit KRAS G12C-dependent signaling and cancer cell growth at sub-micromolar concentrations.

Dendritic Cells but Not Macrophages Sense Tumor Mitochondrial DNA for Cross-priming through Signal Regulatory Protein α Signaling.

  • Xu MM
  • Immunity
  • 2017 Aug 15

Literature context:


Abstract:

Inhibition of cytosolic DNA sensing represents a strategy that tumor cells use for immune evasion, but the underlying mechanisms are unclear. Here we have shown that CD47-signal regulatory protein α (SIRPα) axis dictates the fate of ingested DNA in DCs for immune evasion. Although macrophages were more potent in uptaking tumor DNA, increase of DNA sensing by blocking the interaction of SIRPα with CD47 preferentially occurred in dendritic cells (DCs) but not in macrophages. Mechanistically, CD47 blockade enabled the activation of NADPH oxidase NOX2 in DCs, which in turn inhibited phagosomal acidification and reduced the degradation of tumor mitochondrial DNA (mtDNA) in DCs. mtDNA was recognized by cyclic-GMP-AMP synthase (cGAS) in the DC cytosol, contributing to type I interferon (IFN) production and antitumor adaptive immunity. Thus, our findings have demonstrated how tumor cells inhibit innate sensing in DCs and suggested that the CD47-SIRPα axis is critical for DC-driven antitumor immunity.

Funding information:
  • NCI NIH HHS - R01 CA134563()
  • NCI NIH HHS - R01 CA141975()

Environmental cystine drives glutamine anaplerosis and sensitizes cancer cells to glutaminase inhibition.

  • Muir A
  • Elife
  • 2017 Aug 15

Literature context:


Abstract:

Many mammalian cancer cell lines depend on glutamine as a major tri-carboxylic acid (TCA) cycle anaplerotic substrate to support proliferation. However, some cell lines that depend on glutamine anaplerosis in culture rely less on glutamine catabolism to proliferate in vivo. We sought to understand the environmental differences that cause differential dependence on glutamine for anaplerosis. We find that cells cultured in adult bovine serum, which better reflects nutrients available to cells in vivo, exhibit decreased glutamine catabolism and reduced reliance on glutamine anaplerosis compared to cells cultured in standard tissue culture conditions. We find that levels of a single nutrient, cystine, accounts for the differential dependence on glutamine in these different environmental contexts. Further, we show that cystine levels dictate glutamine dependence via the cystine/glutamate antiporter xCT/SLC7A11. Thus, xCT/SLC7A11 expression, in conjunction with environmental cystine, is necessary and sufficient to increase glutamine catabolism, defining important determinants of glutamine anaplerosis and glutaminase dependence in cancer.

BET Bromodomain Proteins Function as Master Transcription Elongation Factors Independent of CDK9 Recruitment.

  • Winter GE
  • Mol. Cell
  • 2017 Jul 6

Literature context:


Abstract:

Processive elongation of RNA Polymerase II from a proximal promoter paused state is a rate-limiting event in human gene control. A small number of regulatory factors influence transcription elongation on a global scale. Prior research using small-molecule BET bromodomain inhibitors, such as JQ1, linked BRD4 to context-specific elongation at a limited number of genes associated with massive enhancer regions. Here, the mechanistic characterization of an optimized chemical degrader of BET bromodomain proteins, dBET6, led to the unexpected identification of BET proteins as master regulators of global transcription elongation. In contrast to the selective effect of bromodomain inhibition on transcription, BET degradation prompts a collapse of global elongation that phenocopies CDK9 inhibition. Notably, BRD4 loss does not directly affect CDK9 localization. These studies, performed in translational models of T cell leukemia, establish a mechanism-based rationale for the development of BET bromodomain degradation as cancer therapy.

PTEN controls glandular morphogenesis through a juxtamembrane β-Arrestin1/ARHGAP21 scaffolding complex.

  • Javadi A
  • Elife
  • 2017 Jul 27

Literature context:


Abstract:

PTEN controls three-dimensional (3D) glandular morphogenesis by coupling juxtamembrane signaling to mitotic spindle machinery. While molecular mechanisms remain unclear, PTEN interacts through its C2 membrane-binding domain with the scaffold protein β-Arrestin1. Because β-Arrestin1 binds and suppresses the Cdc42 GTPase-activating protein ARHGAP21, we hypothesize that PTEN controls Cdc42 -dependent morphogenic processes through a β-Arrestin1-ARHGAP21 complex. Here, we show that PTEN knockdown (KD) impairs β-Arrestin1 membrane localization, β-Arrestin1-ARHGAP21 interactions, Cdc42 activation, mitotic spindle orientation and 3D glandular morphogenesis. Effects of PTEN deficiency were phenocopied by β-Arrestin1 KD or inhibition of β-Arrestin1-ARHGAP21 interactions. Conversely, silencing of ARHGAP21 enhanced Cdc42 activation and rescued aberrant morphogenic processes of PTEN-deficient cultures. Expression of the PTEN C2 domain mimicked effects of full-length PTEN but a membrane-binding defective mutant of the C2 domain abrogated these properties. Our results show that PTEN controls multicellular assembly through a membrane-associated regulatory protein complex composed of β-Arrestin1, ARHGAP21 and Cdc42.

Wnt-Dependent Inactivation of the Groucho/TLE Co-repressor by the HECT E3 Ubiquitin Ligase Hyd/UBR5.

  • Flack JE
  • Mol. Cell
  • 2017 Jul 20

Literature context:


Abstract:

Extracellular signals are transduced to the cell nucleus by effectors that bind to enhancer complexes to operate transcriptional switches. For example, the Wnt enhanceosome is a multiprotein complex associated with Wnt-responsive enhancers through T cell factors (TCF) and kept silent by Groucho/TLE co-repressors. Wnt-activated β-catenin binds to TCF to overcome this repression, but how it achieves this is unknown. Here, we discover that this process depends on the HECT E3 ubiquitin ligase Hyd/UBR5, which is required for Wnt signal responses in Drosophila and human cell lines downstream of activated Armadillo/β-catenin. We identify Groucho/TLE as a functionally relevant substrate, whose ubiquitylation by UBR5 is induced by Wnt signaling and conferred by β-catenin. Inactivation of TLE by UBR5-dependent ubiquitylation also involves VCP/p97, an AAA ATPase regulating the folding of various cellular substrates including ubiquitylated chromatin proteins. Thus, Groucho/TLE ubiquitylation by Hyd/UBR5 is a key prerequisite that enables Armadillo/β-catenin to activate transcription.

Lck/Hck/Fgr-Mediated Tyrosine Phosphorylation Negatively Regulates TBK1 to Restrain Innate Antiviral Responses.

  • Liu S
  • Cell Host Microbe
  • 2017 Jun 14

Literature context:


Abstract:

Cytosolic nucleic acid sensing elicits interferon production for primary antiviral defense through cascades controlled by protein ubiquitination and Ser/Thr phosphorylation. Here we show that TBK1, a core kinase of antiviral pathways, is inhibited by tyrosine phosphorylation. The Src family kinases (SFKs) Lck, Hck, and Fgr directly phosphorylate TBK1 at Tyr354/394, to prevent TBK1 dimerization and activation. Accordingly, antiviral sensing and resistance were substantially enhanced in Lck/Hck/Fgr triple knockout cells and ectopic expression of Lck/Hck/Fgr dampened the antiviral defense in cells and zebrafish. Small-molecule inhibitors of SFKs, which are conventional anti-tumor therapeutics, enhanced antiviral responses and protected zebrafish and mice from viral attack. Viral infection induced the expression of Lck/Hck/Fgr through TBK1-mediated mobilization of IRF3, thus constituting a negative feedback loop. These findings unveil the negative regulation of TBK1 via tyrosine phosphorylation and the functional integration of SFKs into innate antiviral immunity.

MPI depletion enhances O-GlcNAcylation of p53 and suppresses the Warburg effect.

  • Shtraizent N
  • Elife
  • 2017 Jun 23

Literature context:


Abstract:

Rapid cellular proliferation in early development and cancer depends on glucose metabolism to fuel macromolecule biosynthesis. Metabolic enzymes are presumed regulators of this glycolysis-driven metabolic program, known as the Warburg effect; however, few have been identified. We uncover a previously unappreciated role for Mannose phosphate isomerase (MPI) as a metabolic enzyme required to maintain Warburg metabolism in zebrafish embryos and in both primary and malignant mammalian cells. The functional consequences of MPI loss are striking: glycolysis is blocked and cells die. These phenotypes are caused by induction of p53 and accumulation of the glycolytic intermediate fructose 6-phosphate, leading to engagement of the hexosamine biosynthetic pathway (HBP), increased O-GlcNAcylation, and p53 stabilization. Inhibiting the HBP through genetic and chemical methods reverses p53 stabilization and rescues the Mpi-deficient phenotype. This work provides mechanistic evidence by which MPI loss induces p53, and identifies MPI as a novel regulator of p53 and Warburg metabolism.

Funding information:
  • NIAAA NIH HHS - R01 AA018886()
  • NIDDK NIH HHS - K08 DK101340()
  • NIDDK NIH HHS - P30 DK084567()
  • NIDDK NIH HHS - R01 DK080789()
  • NIDDK NIH HHS - R01 DK099551()
  • NIDDK NIH HHS - R01 DK099558()
  • NIDDK NIH HHS - T32 DK007792()

Mutational phospho-mimicry reveals a regulatory role for the XRCC4 and XLF C-terminal tails in modulating DNA bridging during classical non-homologous end joining.

  • Normanno D
  • Elife
  • 2017 May 13

Literature context:


Abstract:

XRCC4 and DNA Ligase 4 (LIG4) form a tight complex that provides DNA ligase activity for classical non-homologous end joining (the predominant DNA double-strand break repair pathway in higher eukaryotes) and is stimulated by XLF. Independently of LIG4, XLF also associates with XRCC4 to form filaments that bridge DNA. These XRCC4/XLF complexes rapidly load and connect broken DNA, thereby stimulating intermolecular ligation. XRCC4 and XLF both include disordered C-terminal tails that are functionally dispensable in isolation but are phosphorylated in response to DNA damage by DNA-PK and/or ATM. Here we concomitantly modify the tails of XRCC4 and XLF by substituting fourteen previously identified phosphorylation sites with either alanine or aspartate residues. These phospho-blocking and -mimicking mutations impact both the stability and DNA bridging capacity of XRCC4/XLF complexes, but without affecting their ability to stimulate LIG4 activity. Implicit in this finding is that phosphorylation may regulate DNA bridging by XRCC4/XLF filaments.

Funding information:
  • NIAID NIH HHS - R01 AI048758()

RPL10L Is Required for Male Meiotic Division by Compensating for RPL10 during Meiotic Sex Chromosome Inactivation in Mice.

  • Jiang L
  • Curr. Biol.
  • 2017 May 22

Literature context:


Abstract:

The mammalian sex chromosomes have undergone profound changes during their evolution from an ancestral pair of autosomes [1-4]. Specifically, the X chromosome has acquired a paradoxical sex-biased function by redistributing gene contents [5, 6] and has generated a disproportionately high number of retrogenes that are located on autosomes and exhibit male-biased expression patterns [6]. Several selection-based models have been proposed to explain this phenomenon, including a model of sexual antagonism driving X inactivation (SAXI) [6-8] and a compensatory mechanism based on meiotic sex chromosome inactivation (MSCI) [6, 8-11]. However, experimental evidence correlating the function of X-chromosome-derived autosomal retrogenes with evolutionary forces remains limited [12-17]. Here, we show that the deficiency of Rpl10l, a murine autosomal retrogene of Rpl10 with testis-specific expression, disturbs ribosome biogenesis in late-prophase spermatocytes and prohibits the transition from prophase into metaphase of the first meiotic division, resulting in male infertility. Rpl10l expression compensates for the lack of Rpl10, which exhibits a broad expression pattern but is subject to MSCI during spermatogenesis. Importantly, ectopic expression of RPL10L prevents the death of cultured RPL10-deficient somatic cells, and Rpl10l-promoter-driven transgenic expression of Rpl10 in spermatocytes restores spermatogenesis and fertility in Rpl10l-deficient mice. Our results demonstrate that Rpl10l plays an essential role during the meiotic stage of spermatogenesis by compensating for MSCI-mediated transcriptional silencing of Rpl10. These data provide direct evidence for the compensatory hypothesis and add novel insight into the evolution of X-chromosome-derived autosomal retrogenes and their role in male fertility.

Ribonucleotide Reductase Requires Subunit Switching in Hypoxia to Maintain DNA Replication.

  • Foskolou IP
  • Mol. Cell
  • 2017 Apr 20

Literature context:


Abstract:

Cells exposed to hypoxia experience replication stress but do not accumulate DNA damage, suggesting sustained DNA replication. Ribonucleotide reductase (RNR) is the only enzyme capable of de novo synthesis of deoxyribonucleotide triphosphates (dNTPs). However, oxygen is an essential cofactor for mammalian RNR (RRM1/RRM2 and RRM1/RRM2B), leading us to question the source of dNTPs in hypoxia. Here, we show that the RRM1/RRM2B enzyme is capable of retaining activity in hypoxia and therefore is favored over RRM1/RRM2 in order to preserve ongoing replication and avoid the accumulation of DNA damage. We found two distinct mechanisms by which RRM2B maintains hypoxic activity and identified responsible residues in RRM2B. The importance of RRM2B in the response to tumor hypoxia is further illustrated by correlation of its expression with a hypoxic signature in patient samples and its roles in tumor growth and radioresistance. Our data provide mechanistic insight into RNR biology, highlighting RRM2B as a hypoxic-specific, anti-cancer therapeutic target.

Lithocholic Acid Hydroxyamide Destabilizes Cyclin D1 and Induces G0/G1 Arrest by Inhibiting Deubiquitinase USP2a.

  • Magiera K
  • Cell Chem Biol
  • 2017 Apr 20

Literature context:


Abstract:

USP2a is a deubiquitinase responsible for stabilization of cyclin D1, a crucial regulator of cell-cycle progression and a proto-oncoprotein overexpressed in numerous cancer types. Here we report that lithocholic acid (LCA) derivatives are inhibitors of USP proteins, including USP2a. The most potent LCA derivative, LCA hydroxyamide (LCAHA), inhibits USP2a, leading to a significant Akt/GSK3β-independent destabilization of cyclin D1, but does not change the expression of p27. This leads to the defects in cell-cycle progression. As a result, LCAHA inhibits the growth of cyclin D1-expressing, but not cyclin D1-negative cells, independently of the p53 status. We show that LCA derivatives may be considered as future therapeutics for the treatment of cyclin D1-addicted p53-expressing and p53-defective cancer types.

Inhibition of Hematopoietic Cell Kinase Activity Suppresses Myeloid Cell-Mediated Colon Cancer Progression.

  • Poh AR
  • Cancer Cell
  • 2017 Apr 10

Literature context:


Abstract:

Aberrant activation of the SRC family kinase hematopoietic cell kinase (HCK) triggers hematological malignancies as a tumor cell-intrinsic oncogene. Here we find that high HCK levels correlate with reduced survival of colorectal cancer patients. Likewise, increased Hck activity in mice promotes the growth of endogenous colonic malignancies and of human colorectal cancer cell xenografts. Furthermore, tumor-associated macrophages of the corresponding tumors show a pronounced alternatively activated endotype, which occurs independently of mature lymphocytes or of Stat6-dependent Th2 cytokine signaling. Accordingly, pharmacological inhibition or genetic reduction of Hck activity suppresses alternative activation of tumor-associated macrophages and the growth of colon cancer xenografts. Thus, Hck may serve as a promising therapeutic target for solid malignancies.

Funding information:
  • NIAID NIH HHS - R01 AI065495()
  • NIAID NIH HHS - R01 AI068150()

ZNF598 and RACK1 Regulate Mammalian Ribosome-Associated Quality Control Function by Mediating Regulatory 40S Ribosomal Ubiquitylation.

  • Sundaramoorthy E
  • Mol. Cell
  • 2017 Feb 16

Literature context:


Abstract:

Ribosomes that experience terminal stalls during translation are resolved by ribosome-associated quality control (QC) pathways that oversee mRNA and nascent chain destruction and recycle ribosomal subunits. The proximal factors that sense stalled ribosomes and initiate mammalian ribosome-associated QC events remain undefined. We demonstrate that the ZNF598 ubiquitin ligase and the 40S ribosomal protein RACK1 help to resolve poly(A)-induced stalled ribosomes. They accomplish this by regulating distinct and overlapping regulatory 40S ribosomal ubiquitylation events. ZNF598 primarily mediates regulatory ubiquitylation of RPS10 and RPS20, whereas RACK1 regulates RPS2, RPS3, and RPS20 ubiquitylation. Gain or loss of ZNF598 function or mutations that block RPS10 or RPS20 ubiquitylation result in defective resolution of stalled ribosomes and subsequent readthrough of poly(A)-containing stall sequences. Together, our results indicate that ZNF598, RACK1, and 40S regulatory ubiquitylation plays a pivotal role in mammalian ribosome-associated QC pathways.

Funding information:
  • NIGMS NIH HHS - DP2 GM119132()
  • NIGMS NIH HHS - P50 GM085764()

Caspase-8 Acts in a Non-enzymatic Role as a Scaffold for Assembly of a Pro-inflammatory "FADDosome" Complex upon TRAIL Stimulation.

  • Henry CM
  • Mol. Cell
  • 2017 Feb 16

Literature context:


Abstract:

TRAIL is a potent inducer of apoptosis and has been studied almost exclusively in this context. However, TRAIL can also induce NFκB-dependent expression of multiple pro-inflammatory cytokines and chemokines. Surprisingly, whereas inhibition of caspase activity blocked TRAIL-induced apoptosis, but not cytokine production, knock down or deletion of caspase-8 suppressed both outcomes, suggesting that caspase-8 participates in TRAIL-induced inflammatory signaling in a scaffold role. Consistent with this, introduction of a catalytically inactive caspase-8 mutant into CASP-8 null cells restored TRAIL-induced cytokine production, but not cell death. Furthermore, affinity precipitation of the native TRAIL receptor complex revealed that pro-caspase-8 was required for recruitment of RIPK1, via FADD, to promote NFκB activation and pro-inflammatory cytokine production downstream. Thus, caspase-8 can serve in two distinct roles in response to TRAIL receptor engagement, as a scaffold for assembly of a Caspase-8-FADD-RIPK1 "FADDosome" complex, leading to NFκB-dependent inflammation, or as a protease that promotes apoptosis.

Funding information:
  • Worldwide Cancer Research - 14-0323()

Single-Molecule Analysis of mtDNA Replication Uncovers the Basis of the Common Deletion.

  • Phillips AF
  • Mol. Cell
  • 2017 Feb 2

Literature context:


Abstract:

Mutations in mtDNA lead to muscular and neurological diseases and are linked to aging. The most frequent aberrancy is the "common deletion" that involves a 4,977-bp region flanked by 13-bp repeats. To investigate the basis of this deletion, we developed a single-molecule mtDNA combing method. The analysis of replicating mtDNA molecules provided in vivo evidence in support of the asymmetric mode of replication. Furthermore, we observed frequent fork stalling at the junction of the common deletion, suggesting that impaired replication triggers the formation of this toxic lesion. In parallel experiments, we employed mito-TALENs to induce breaks in distinct loci of the mitochondrial genome and found that breaks adjacent to the 5' repeat trigger the common deletion. Interestingly, this process was mediated by the mitochondrial replisome independent of canonical DSB repair. Altogether, our data underscore a unique replication-dependent repair pathway that leads to the mitochondrial common deletion.

Computationally designed high specificity inhibitors delineate the roles of BCL2 family proteins in cancer.

  • Berger S
  • Elife
  • 2016 Nov 2

Literature context:


Abstract:

Many cancers overexpress one or more of the six human pro-survival BCL2 family proteins to evade apoptosis. To determine which BCL2 protein or proteins block apoptosis in different cancers, we computationally designed three-helix bundle protein inhibitors specific for each BCL2 pro-survival protein. Following in vitro optimization, each inhibitor binds its target with high picomolar to low nanomolar affinity and at least 300-fold specificity. Expression of the designed inhibitors in human cancer cell lines revealed unique dependencies on BCL2 proteins for survival which could not be inferred from other BCL2 profiling methods. Our results show that designed inhibitors can be generated for each member of a closely-knit protein family to probe the importance of specific protein-protein interactions in complex biological processes.

Funding information:
  • NIDDK NIH HHS - R21 DK106584(United States)

TP53 drives invasion through expression of its Δ133p53β variant.

  • Gadea G
  • Elife
  • 2016 Sep 15

Literature context:


Abstract:

TP53 is conventionally thought to prevent cancer formation and progression to metastasis, while mutant TP53 has transforming activities. However, in the clinic, TP53 mutation status does not accurately predict cancer progression. Here we report, based on clinical analysis corroborated with experimental data, that the p53 isoform Δ133p53β promotes cancer cell invasion, regardless of TP53 mutation status. Δ133p53β increases risk of cancer recurrence and death in breast cancer patients. Furthermore Δ133p53β is critical to define invasiveness in a panel of breast and colon cell lines, expressing WT or mutant TP53. Endogenous mutant Δ133p53β depletion prevents invasiveness without affecting mutant full-length p53 protein expression. Mechanistically WT and mutant Δ133p53β induces EMT. Our findings provide explanations to 2 long-lasting and important clinical conundrums: how WT TP53 can promote cancer cell invasion and reciprocally why mutant TP53 gene does not systematically induce cancer progression.

Inactivation of oncogenic cAMP-specific phosphodiesterase 4D by miR-139-5p in response to p53 activation.

  • Cao B
  • Elife
  • 2016 Jul 7

Literature context:


Abstract:

Increasing evidence highlights the important roles of microRNAs in mediating p53's tumor suppression functions. Here, we report miR-139-5p as another new p53 microRNA target. p53 induced the transcription of miR-139-5p, which in turn suppressed the protein levels of phosphodiesterase 4D (PDE4D), an oncogenic protein involved in multiple tumor promoting processes. Knockdown of p53 reversed these effects. Also, overexpression of miR-139-5p decreased PDE4D levels and increased cellular cAMP levels, leading to BIM-mediated cell growth arrest. Furthermore, our analysis of human colorectal tumor specimens revealed significant inverse correlation between the expression of miR-139-5p and that of PDE4D. Finally, overexpression of miR-139-5p suppressed the growth of xenograft tumors, accompanied by decrease in PDE4D and increase in BIM. These results demonstrate that p53 inactivates oncogenic PDE4D by inducing the expression of miR-139-5p.