An Upstream Open Reading Frame in Phosphatase and Tensin Homolog Encodes a Circuit Breaker of Lactate Metabolism

•MP31 is a micropeptide encoded from 5? UTR of PTEN•MP31 localizes in mitochondria and limits lactate-pyruvate conversion•MP31 loss promotes lactate metabolism OXPHOS glioblastoma•Recombinant MP31 has therapeutic effects on GBM mice model without inducing toxicity The metabolic role micropeptides generated untranslated regions remains unclear. Here we describe MP31, by the upstream open reading frame (uORF) phosphatase tensin homolog (PTEN) acting as “circuit breaker” that conversion competing with mitochondrial dehydrogenase (mLDH) for nicotinamide adenine dinucleotide (NAD+). Knocking out enhanced global metabolism, manifesting accelerated oxidative phosphorylation (OXPHOS) increased consumption production. Conditional knockout (cKO) mouse astrocytes initiated gliomagenesis shortened overall survival animals, establishing tumor-suppressing MP31. Recombinant administered intraperitoneally penetrated blood-brain barrier inhibited xenografts neurological toxicity, suggesting clinical implication application this micropeptide. Our findings reveal novel mode MP31-orchestrated reprogramming glioblastoma. Even radical resection plus radiation thermotherapy, rapid cancer recurrence inevitable, prognosis dismal glioblastoma (GBM) patients (Wen Kesari, 2008Wen P.Y. Kesari S. Malignant gliomas adults.N. Engl. J. Med. 2008; 359: 492-507Crossref PubMed Scopus (3028) Google Scholar). highly heterogenetic tumor (Brennan et al., 2013Brennan C.W. Verhaak R.G. McKenna A. Campos B. Noushmehr H. Salama S.R. Zheng Chakravarty D. Sanborn J.Z. Berman S.H. al.TCGA Research NetworkThe somatic genomic landscape glioblastoma.Cell. 2013; 155: 462-477Abstract Full Text PDF (2529) From perspective cells could be divided into quiescent rapidly proliferating (Shi 2019Shi Y. Lim S.K. Liang Q. Iyer S.V. Wang H.Y. Z. Xie X. Sun Chen Y.J. Tabar V. al.Gboxin an inhibitor targets glioblastoma.Nature. 2019; 567: 341-346Crossref (87) Scholar; Shibao 2018Shibao Minami N. Koike Fukui Yoshida K. Saya Sampetrean O. Metabolic heterogeneity plasticity glioma stem model.Neuro-oncol. 2018; 20: 343-354Crossref (29) To date, stemness property are considered primary reason tumorigenesis, resistance, (Chen 2012Chen McKay R.M. Parada L.F. glioma: lessons genomics, models, cells.Cell. 2012; 149: 36-47Abstract (372) Although there no available strategies target these slow-cycling cells, their distinct biological features have drawn attention. Among prominent differences, proliferative prefer aerobic glycolysis (the Warburg effect), whereas utilize energy source (Hoang-Minh 2018Hoang-Minh L.B. Siebzehnrubl F.A. Yang C. Suzuki-Hatano Dajac Loche T. Andrews Schmoll Massari M. Patel Amin al.Infiltrative drug-resistant support glioblastoma.EMBO 37: e98772Crossref (43) Kuntz 2017Kuntz E.M. Baquero P. Michie A.M. Dunn Tardito Holyoake T.L. Helgason G.V. Gottlieb E. Targeting eradicates therapy-resistant chronic myeloid leukemia cells.Nat. 2017; 23: 1234-1240Crossref (195) indivisible link between effect been subject profound reappraisal recent years owing to extensive studies i.e., intact or even stronger respiration many (Vyas 2016Vyas Zaganjor Haigis M.C. Mitochondria cancer.Cell. 2016; 166: 555-566Abstract (700) Scholar), undefined purpose (Corbet Feron, 2017Corbet Feron Cancer cell mitochondria: nutrient TCA cycle fueling.Biochim Biophys Acta Rev Cancer. 1868: 7-15Crossref (72) controversial carboxylates dominant fuel cancers vivo (Faubert 2017Faubert Li K.Y. Cai L. Hensley C.T. Kim Zacharias L.G. Do Q.N. Doucette Burguete al.Lactate human lung tumors.Cell. 171: 358-371.e9Abstract (474) Hui 2017Hui Ghergurovich J.M. Morscher R.J. Jang Teng Lu W. Esparza L.A. Reya Le Zhan Yanxiang Guo al.Glucose feeds via circulating lactate.Nature. 551: 115-118Crossref (525) Efforts advance discussion pointed lactate, final product produced pyruvate (LDH). Lactate traditionally “dead end” metabolite. However, can recycle NAD+ reduced NADH during glycolysis, excretion may maintain pH homeostasis inside acidify extracellular space (Brand 2016Brand Singer Koehl G.E. Kolitzus Schoenhammer G. Thiel Matos Bruss Klobuch Peter al.LDHA-associated lactic acid production blunts immunosurveillance T NK cells.Cell Metab. 24: 657-671Abstract (534) 2014Xie Hanai Ren J.G. Kats Burgess Bhargava Signoretti Billiard Duffy K.J. Grant al.Targeting dehydrogenase--a inhibits tumorigenesis progression models impacts tumor-initiating 2014; 19: 795-809Abstract (275) Furthermore, converted carboxylate contributes predominates over (San-Millán Brooks, 2017San-Millán I. Brooks G.A. Reexamining metabolism: carcinogenesis explanation effect.Carcinogenesis. 38: 119-133PubMed These corroborate symbiosis which shuttled glycolytic environment (fast-cycling stroma, fibroblasts) mitochondrion-respiration-dependent cells. mechanistic insight preferred utilization elusive. Emerging evidence shows small frames (sORFs) at atypical locations, such UTR, 3? noncoding RNAs (ncRNAs), create functional peptides (Bi 2017Bi Ramirez-Martinez Cannavino McAnally J.R. Shelton Sánchez-Ortiz Bassel-Duby R. Olson E.N. Control muscle formation fusogenic myomixer.Science. 356: 323-327Crossref (156) Magny 2013Magny E.G. Pueyo J.I. Pearl F.M. Cespedes M.A. Niven J.E. Bishop S.A. Couso J.P. Conserved regulation cardiac calcium uptake frames.Science. 341: 1116-1120Crossref (199) sORF-encoded reported pervasively modulate translation (Andrews Rothnagel, 2014Andrews S.J. Rothnagel J.A. short frames.Nat. Rev. Genet. 15: 193-204Crossref (317) Interestingly, some sORFs long intergenic (lincRNAs) physiological roles cellular respiration, fatty oxidation adequately demonstrated (Chugunova 2019Chugunova Loseva Mazin Mitina Navalayeu Bilan Vishnyakova Marey Golovina Serebryakova al.LINC00116 codes peptide linking lipid metabolism.Proc. Natl. Acad. Sci. USA. 116: 4940-4945Crossref (36) Makarewich 2018Makarewich C.A. Baskin K.K. Munir A.Z. Bezprozvannaya Sharma Khemtong Shah Malloy C.R. Szweda L.I. al.MOXI enhances ?-oxidation.Cell Rep. 3701-3709Abstract (61) Stein 2018Stein C.S. Jadiya Zhang McLendon Abouassaly G.M. Witmer N.H. Anderson E.J. Elrod J.W. Boudreau R.L. Mitoregulin: lncRNA-encoded microprotein supports supercomplexes respiratory efficiency.Cell 3710-3720.e8Abstract (80) discoveries regulators sORFs, particularly aberrant hold promise cancer. In current study, sought identify gene locations investigate previously unknown key might serve potential treatment. We specifically described identification uORF PTEN. then functionally validated importance its implications. differentially translated new performed ribosome profiling 10 specimens paired normal brain tissues (NBs) adjacent tumors, (Figure S1A) (Ingolia 2012Ingolia N.T. Brar Rouskin McGeachy Weissman J.S. strategy monitoring deep sequencing ribosome-protected mRNA fragments.Nat. Protoc. 7: 1534-1550Crossref (623) Most fragments (RPFs) (86.14%) were derived main (mORFs) known protein-coding genes, remaining RPFs mapped transcripts annotated regions, including (5.15%) (2.28%) genes lincRNAs (6.43%) S1B). compared relative abundance cancerous (Table S1). GBM, RPF was lower than (p < 0.05), while significant differences observed lincRNAs, tend shorter UTRs through alternative polyadenylation S1C) (Xia 2014Xia Donehower Cooper T.A. Neilson Wheeler D.A. Wagner Dynamic analyses RNA-seq 3?-UTR across seven tumour types.Nat. Commun. 5: 5274Crossref (217) Metagene reads revealed most 29- 30-nt in-frame ends (position 1 codon P-site, offset +12 nt) (Data S1) showed 3-nucleotide periodicity S2), high quality ribosome-profiling data (Calviello Ohler, 2017Calviello Ohler U. Beyond read-counts: ribo-seq analysis understand functions transcriptome.Trends 33: 728-744Abstract (49) next analyzed differential (DTR) NB every single ORF genome-wide level. addition mORFs also searched noncanonical using ATG initiation transcript sequences uORFs, downstream ORFs (dORFs), (lincORFs). level each quantified based RPFs. signals (average read count ? 1) retained, 14,808 mORFs, 3,061 6,329 dORFs, 5,764 lincORFs. Differential further conducted, 1,556 DTR-mORFs, 140 DTR-uORFs, 549 DTR-dORFs, 118 DTR-lincORFs identified 1A; Table S2). Peptides uORFs regulate (Calvo 2009Calvo S.E. Pagliarini D.J. Mootha V.K. Upstream cause widespread reduction protein expression polymorphic among humans.Proc. 2009; 106: 7507-7512Crossref (504) Sendoel 2017Sendoel Rodriguez E.H. Naik Gomez N.C. Hurwitz Levorse Dill B.D. Schramek Molina al.Translation unconventional start sites drives initiation.Nature. 541: 494-499Crossref (159) explored 2,983 uORF-mORF pairs detectable both corresponding mORFs). positive correlation (r2 = 0.59). variations samples classified five groups. A total 303 did not show changes, exhibited changes. Fifty-eight changes mORF Eighty-two homo-directional (both upregulated downregulated). total, 2,540 changed samples. observe any opposite change patterns 1B). Noncanonical bound ribosomes necessarily translate actively (Guttman 2013Guttman Russell Ingolia Lander E.S. Ribosome provides large do encode proteins.Cell. 154: 240-251Abstract find active translation, calculated release score (RRS) modified By comparing RRS scores 194 18 expressed 1C). 6 samples, other 12 downregulated, respective levels 1D). noticed putative uORF, PTEN_31AA, located PTEN, one critical tumor-suppressive GBM. PTEN CDS, PTEN-long (Hopkins 2013Hopkins Fine Steinbach Dendy Rapp Shaw Pappas Yu Hodakoski Mense al.A secreted enters alter signaling survival.Science. 399-402Crossref (207) Scholar) PTEN-? (Liang 2017Liang Yin Ruan Zhao McNutt PTEN? alternatively isoform regulates rDNA transcription.Nat. 8: 14771Crossref (56) 1E, upper; S3). occupancy PTEN_31AA downregulated significantly (log2 fold ?1.55, FDR 0.013). biochemical characteristics, implying fully defined, especially variants CDS. Inspection RNA bp ?122 ?217 predicted 31-amino termed conservative N-terminal different species lower). authenticate CDS compromised (MP31 KO), Flag-tag knocked immediately stop (Flag KI) 293T (Figures S2A S2B). An monoclonal antibody S2C). Immunoblotting (IB) anti-MP31 anti-Flag suggested indeed additional mass spectrometry (MS) obtained 1F S2D). Notably, KO affect expression. decreased NB. Using IB, verified multiple patient-derived neural (NSCs) (NHAs) several randomly selected 1G). often co-deleted co-expressed tissue but established lines S2E). After confirming specificity immunofluorescence (IF) S2F), examined cohort 107 remarkably it function 1H; n 107). Based irrelevance expression, First, localization determined stably overexpressing MP31-GFP endogenously NSCs, 2A S3A). clear observed. Further fractionation experiments MP31-overexpressing NSCs indicated near inner membrane matrix 2B). Novel components (Cao 2016Cao L.L. Riascos-Bernal D.F. Chinnasamy Dunaway C.M. Hou Pujato O’Rourke B.P. Miskolci Hodgson al.Control growth cadherin Fat1.Nature. 539: 575-578Crossref (25) Kory 2018Kory Wyant Prakash Uit de Bos Bottanelli F. Pacold M.E. Chan Lewis Keys H.R. al.SFXN1 serine transporter required one-carbon metabolism.Science. 362: eaat9528Crossref (70) vivo, MP35 (a MP31)-KO CRISPR/Cas9 technology S3B–S3D). mice, elevated glucose (positron-emitting 18F-fluorodeoxyglucose high-resolution positron emission tomography/computed tomography, 18F-FDG-PET/CT) 2C), S3E S3F), ATP (serum concentration; Figure 2D), perigonadal fat pad weight fat/body ratio 2E, 2F, S3G). Administration 1-month high-fat diet (HFD) less effectively induced hepatic adipose infiltration MP35-KO wild-type S3H). metabonomic glycol-metabolism, supplies 70% body energy, embryonic fibroblasts (MEFWT MEFKO). Compared MEFWT, MEFKO substrates S4A, left). oxygen rate (OCR) explain higher right). Sequentially, concentration MEFWT whether glycol tested 4121 456 MP31-KO (NSCKO) control Metabonomic overexpression attenuated substrate 3A S4B, left), OCR populations 3B, 3C, contrast, NSCKO NSCWT 3D–3F, S4C, S4D). This suggests uncharacterized regulator controls mainly restricting OXPHOS. assay, impaired sphere limited dilution assay (LDA) viability 3G S4E). proliferation rates 3H). Stably tumorigenicity associated prolonged harboring intracranial 3I S4F). GFAPCre (Zhuo 2001Zhuo Theis Alvarez-Maya Brenner Willecke Messing hGFAP-cre transgenic manipulation glial neuronal vivo.Genesis. 2001; 31: 85-94Crossref (460) Scholar)-induced conditional S5A–S5C) symptoms included seizure, ataxia, and/or paralysis 55–65 weeks age 3J, Histopathologically, 17 symptomatic presented phenotype, tumors lineage progenitor markers Nestin GFAP Ki67 collectively mice. Given affects enzymes. Neither nor altered kinase M1/2 (PKM1/2), A/B (LDHA/B), hexokinase 2 (HK2), phosphofructokinase (PFK), phosphoglycerate (PGK1), oxoglutarate (2-OGDH) S5D). immunoprecipitation (IP) binding partners. 283 proteins immunoprecipitants pulled down S4). Eighteen Gene Ontology (GO) terms component category related enriched candidate proteins, “mitochondrion” (58 associated) “mitochondrial part” (43 4A). Venn diagram 43 part localized 4B). pathway enrichment conducted 58 “mitochondrion-associated” proteins. Pathways “oxidative phosphorylation” “the electron transport” 4C). enzyme candidates LDHBs activity screen. mutual interaction LDH confirmed IP 4D S5E). As LDHB predominantly 2016Chen Mahieu N.G. Huang Singh Crawford P.A. Johnson S.L. Gross R.W. Schaefer Patti G.J. mammalian mitochondria.Nat. Chem. Biol. 12: 937-943Crossref (117) ascertained LDHB/MP31 NSCs. endogenous colocalized 4E). Mitochondrial shown play central converting pyruvate, differs cytosolic (cLDH) Kane, 2014Kane lactate-malate-aspartate shuttle work.Front. Neurosci. 366Crossref (53) found mLDH 4F, lanes 4; S5F, catalyzes consuming NAD+, generating H+ outside matrix. cross-intermembrane coupled concentrations promote transport Indeed, 4G specific knockdown abolished activity, potential, more NSCKO, indicating MP31/LDHB connection 4F–4H S5G). Patient-derived manifested S5H), supporting theory aberrantly activated malignancies (Brisson 2016Brisson Ba?ski Sboarina Dethier Danhier Fontenille M.J. Van Hée V.F. Vazeille Tardy Falces B lysosome autophagy cancer.Cancer Cell. 30: 418-431Abstract (85) cross-membrane 4I–4K S5I–S5L). partially rescued (reduced production) (OGDH) OCR, measured OGDH NSC MP31-OV S5M). results exerted inhibiting mLDH-mediated conversion. demonstrate interacts function, molecular docking simulation LDHA/B. R3, S5, Y12, R21 residues involved D55, R98, K56, Q100 LDHA salt bridges hydrogen bond interactions. addition, namely, A13, R19, R21, interacted residues, Q101, R99, D52, D56, E102, interactions S5). LDHA/B S5 Y12 prevented interacting K56 R99 D52 LDHB, all amino 5A 5B ) (Cameron 2004Cameron Read Tranter Winter V.J.