LncRNA NBR2 engages a metabolic checkpoint by regulating AMPK under energy stress

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1 A RT I C L E S LncRNA NBR engges metolic checkpoint y regulting under energy stress Xiowen Liu 1,8, Zhen-Dong Xio 1,8, Leng Hn, Jiexin Zhng 3, Szu-Wei Lee,5, Wenqi Wng 1, Hyemin Lee 1, Li Zhung 1, Junjie Chen 1,5, Hui-Kun Lin,5,6,7, Jing Wng 3, Hn Ling 3 nd Boyi Gn 1,,5,9 Long non-coding RNAs (lncrnas) hve emerged s criticl regultors in vrious cellulr processes. However, the potentil involvement of lncrnas in kinse signlling remins lrgely unknown. AMP-ctivted protein kinse () cts s criticl sensor of cellulr energy sttus. Here we show tht the lncrna NBR (neighour of BRCA1 gene ) is induced y the LKB1 pthwy under energy stress. On energy stress, NBR in turn intercts with nd promotes kinse ctivity, thus forming feed-forwrd loop to potentite ctivtion during energy stress. Depletion of NBR ttenutes energy-stress-induced ctivtion, resulting in unchecked cell cycling, ltered poptosis/utophgy response, nd incresed tumour development in vivo. NBR is downregulted nd its low expression correltes with poor clinicl outcomes in some humn cncers. Together, the results of our study uncover mechnism coupling lncrnas with metolic stress response, nd provides rod frmework to understnd further the regultion of kinse signlling y lncrnas. Mmmlin genomes encode more thn 1, long non-coding RNAs (lncrnas), RNA molecules tht re longer thn nucleotides nd do not seem to encode proteins 1,. Although lncrnas were trditionlly viewed s the products tht re generted from the ckground noise of trnscription nd thus exert little fitness dvntge to the cells, it hs ecome incresingly cler tht these lncrnas ply importnt iologicl functions, nd their dysregultion hs een connected to vrious humn diseses, including cncer 3 6. Most current studies focus on lncrna function in the nucleus, prtly ecuse most of the est-understood lncrnas, such s XIST (ref. 7), HOTAIR (ref. 8), HOTTIP (ref. 9), re ll chromtin-ssocited lncrnas, which re minly loclized in the nucleus. These studies hve illustrted diverse rnge of functions of lncrnas in the regultion of chromtin sttus, trnscription nd RNA processing, mong others 1,1. Mny lncrnas hve lso een identified in the cytosol 11. In fct, it hs een suggested tht most lncrnas proly spend most of their lifetime in the cytoplsm 1. However, the exct functions of cytoplsmic loclized lncrnas, prticulrly their potentil functions in the regultion of kinse signlling in the cytoplsm, remin poorly understood. In ddition, lthough lncrnas hve een shown to regulte diverse iologicl processes, the role of lncrnas in mediting metolic checkpoint remins lrgely unexplored. The AMP-ctivted protein kinse () serves s criticl sensor of cellulr energy sttus nd is ctivted under energy stress conditions with n incresed cellulr AMP/ATP rtio 1. AMP inding to nd susequent phosphoryltion t Thr17 y the upstrem kinse LKB1 leds to ctivtion Activted then phosphoryltes numer of downstrem trgets to inctivte ATP-consuming nolic processes nd to ctivte ATPgenerting ctolic processes 16. Thus, minly functions s metolic checkpoint to restore energy lnce in response to energy stress. One mjor nolic process inhiited y in response to energy stress is mmmlin trget of rpmycin complex 1 (mtorc1)-medited protein synthesis nd cell growth 17. In response to energy stress, inctivtes mtorc1 nd represses protein synthesis through phosphoryltion of Rptor, component of mtorc1, nd the TSC1 TSC complex, negtive regultor of mtorc1 (refs 18,19). lso functions to promote utophgy nd cell survivl under energy stress through its phosphoryltion of utophgy regultors, such s ULK1 (refs,1). As nolic 1 Deprtment of Experimentl Rdition Oncology, The University of Texs MD Anderson Cncer Center, 1515 Holcome Boulevrd, Houston, Texs 773, USA. Deprtment of Biochemistry nd Moleculr Biology, The University of Texs Helth Science Center t Houston McGovern Medicl School, Houston, Texs 773, USA. 3 Deprtment of Bioinformtics nd Computtionl Biology, The University of Texs MD Anderson Cncer Center, 1515 Holcome Boulevrd, Houston, Texs 773, USA. Deprtment of Moleculr nd Cellulr Oncology, The University of Texs MD Anderson Cncer Center, 1515 Holcome Boulevrd, Houston, Texs 773, USA. 5 Progrm of Genes nd Development, nd Progrm of Cncer Biology, The University of Texs Grdute School of Biomedicl Sciences, 1515 Holcome Boulevrd, Houston, Texs 773, USA. 6 Deprtment of Cncer Biology, Wke Forest School of Medicine, Winston-Slem, North Crolin 7157, USA. 7 Grdute Institute of Bsic Medicl Science, Chin Medicl University, Tichung, Tiwn. 8 These uthors contriuted eqully to this work. 9 Correspondence should e ddressed to B.G. (e-mil: gn@mdnderson.org) Received Ferury 15; ccepted 1 Ferury 16; pulished online 1 Mrch 16; DOI: 1.138/nc338 NATURE CELL BIOLOGY VOLUME 18 NUMBER APRIL Mcmilln Pulishers Limited. All rights reserved

2 A RT I C L E S Reltive NBR levels 8 6 mm glucose mm glucose P =.5 1 P = P = P =. 1 3 P = P = P = Reltive NBR levels P = P = P = 1. 1 P = 3. 1 mm DG 5 mm DG P =. 1 P = P = O SLR MDA-MB-31 BT-59 MCF-7 HEK-93T DU15 A59 HeL 786-O SLR MDA-MB-31 BT-59 MCF-7 HEK-93T DU15 A59 HeL c Reltive NBR levels HeL P =.8 1 A59 mm glucose mm glucose 7 6 mm glucose mm glucose EV LKB1 EV LKB1 Reltive NBR levels P =. 1 5 d HeL Glucose EV LKB1 (mm) p- p-acc LKB1 GAPDH A59 EV LKB1 M r (K) e P =. 1 3 f P = g P = Reltive NBR levels Reltive NBR levels Reltive NBR levels Control A76966 Compound C ( µm) Glucose ( mm) sirna + + Glucose ( mm) + + Figure 1 Energy stress induces NBR expression through the LKB1 pthwy. (,) Vrious cell lines were cultured in or mm glucosecontining medium (), or or 5 mm DG-contining medium () for 1 h, nd then sujected to rel-time PCR nlysis to mesure NBR expression (men ± s.d., n = 3 iologiclly independent extrcts, twotiled pired Student s t-test). (c,d) HeL or A59 cells stly expressing empty vector (EV) or LKB1 expression vectors were cultured in or mm glucose-contining medium, nd then sujected to rel-time PCR (c) (men ± s.d., n = 3 iologiclly independent extrcts, two-tiled pired Student s t-test) nd western lotting nlyses (d). (e) MDA-MB-31 cells treted with 1 µm A76966 were sujected to rel-time PCR nlysis to mesure NBR expression (men ± s.d., n=3 iologiclly independent extrcts, two-tiled pired Student s t-test). (f) MDA-MB-31 cells were treted with µm compound C in or mm glucose-contining medium for h, nd then sujected to rel-time PCR nlysis to mesure NBR expression (men ± s.d., n = 3 iologiclly independent extrcts, two-tiled pired Student s t-test). (g) MDA-MB-31 cells trnsfected with α or control (Ctrl) sirna were cultured in or mm glucose-contining medium for h, nd then sujected to reltime PCR nlysis to mesure NBR expression (men ± s.d., n=3 iologiclly independent extrcts, two-tiled pired Student s t-test). Source dt for c,e g cn e found in Supplementry Tle 1. Unprocessed originl scns of lots re shown in Supplementry Fig. 8. processes, such s protein nd lipid synthesis, often exert progrowth effects in tumour development, it is well documented tht ctivtion serves to inhiit tumour development in mny cncers. Consistent with this, oth the upstrem kinse LKB1 nd downstrem effectors of, such s TSC1 nd TSC, re on fide tumour suppressors nd re mutted in hmrtom tumour syndromes nd vrious spordic cncers 3. Although the iologicl functions of nd its downstrem effectors 3 16 Mcmilln Pulishers Limited. All rights reserved NATURE CELL BIOLOGY VOLUME 18 NUMBER APRIL 16

3 A RT I C L E S Reltive NBR level O P =.9 1 Ctrl shrna NBR NBR shrna1 shrna Ctrl NBR NBR Glucose shrna shrna1 shrna (mm) p- p-acc ACC p-rptor Rptor 786-O Ctrl NBR NBR shrna shrna1 shrna Ctrl shrna NBR shrna1 MDA-MB-31 NBR shrna M r (K) 1 1 Reltive NBR level MDA-MB-31 P =.1 1 Ctrl shrna NBR shrna1 NBR shrna c Ctrl NBR NBR Glucose shrna shrna1 shrna (mm) p-s6k S6K p-s6 S6 786-O Ctrl NBR NBR shrna shrna1 shrna Ctrl NBR NBR shrna shrna1 shrna MDA-MB-31 M r (K) d Ctrl NBR NBR Ctrl NBR NBR e Ctrl NBR NBR shrna shrna1 shrna shrna shrna1 shrna shrna shrna1 shrna DG (5 mm) A M p- r (K) p- p-acc ACC p-rptor 1 p-acc ACC M r (K) Rptor 1 p-s6 p-s6k S6K S6 MDA-MB-31 p-s6 S6 786-O MDA-MB-31 Figure NBR regultes mtorc1 signlling under energy stress. () Br grph showing NBR-shRNA-medited knockdown efficiency y reltime PCR nlysis in 786-O nd MDA-MB-31 cells (men ± s.d., n=3 iologiclly independent extrcts, two-tiled pired Student s t-test). (,c) 786-O or MDA-MB-31 cells infected with either control shrna or NBR shrna were cultured in medium with different concentrtions of glucose for h. Cell lystes were then nlysed y western lotting. (d) 786-O or MDA-MB-31 cells infected with either control shrna or NBR shrna were cultured in or 5 mm DG-contining medium for 1 (for MDA-MB-31 cells) or 16 (for 786-O cells) h. Cell lystes were then nlysed y western lotting. (e) MDA-MB-31 cells infected with either control shrna or NBR shrna were cultured in or 1 µm A76966-contining medium for 1 h. Cell lystes were then nlysed y western lotting. Source dt for cn e found in Supplementry Tle 1. Unprocessed originl scns of lots re shown in Supplementry Fig. 8. involved in cncer development hve een extensively studied,6, the regultory mechnisms of ctivtion y energy stress remin incompletely understood. In prticulr, it remins completely unknown whether ny lncrna is involved in the -medited metolic checkpoint. In this study, we identify neighour of BRCA1 gene (NBR) s n energy-stress-induced lncrna nd show tht NBR intercts with nd potentites ctivtion under energy stress. Consistent with the tumour suppression function of, NBR deficiency promotes unchecked cell cycling under energy stress nd enhnces tumour development in vivo, nd NBR is downregulted in humn cncers. Our study thus revels previously unpprecited regultory mechnism y lncrnas to regulte kinse function nd to medite cellulr energy responses. NATURE CELL BIOLOGY VOLUME 18 NUMBER APRIL Mcmilln Pulishers Limited. All rights reserved 33

4 A RT I C L E S RESULTS Energy stress induces NBR expression through the LKB1 pthwy To identify energy-stress-induced lncrnas, we conducted n RNA sequencing experiment in 786-O cells tht hd een cultured in glucose-contining or glucose-free medium. Susequent computtionl nlysis identified NBR s one of the long intergenic non-coding RNAs (lincrnas) induced y glucose strvtion. The NBR gene encodes different splicing isoforms rnging from 1 to kiloses (Supplementry Fig. 1). It hs een shown tht NBR is expressed in most of the tissues exmined 7. However, the NBR gene does not seem to encode protein, nd its potentil function remins unknown. Rel-time PCR reveled tht glucose strvtion induced NBR expression in different cncer cell lines, except HeL nd A59 cells, which re LKB1 deficient (Fig. 1). Tretment with the glucose nlogue -deoxy-glucose (DG), nother energy stress inducer tht inhiits hexokinse nd locks glycolysis, yielded similr results (Fig. 1). Importntly, re-expression of LKB1 in these LKB1-deficient cells restored energy-stress-induced NBR expression (Fig. 1c,d). In ddition, tretment with A76966 (n ctivtor) induced NBR expression (Fig. 1e), wheres inctivtion y compound C (n inhiitor) tretment or sirna-medited α knockdown significntly ttenuted glucose strvtion-induced NBR expression (Fig. 1f,g nd Supplementry Fig. ). Together, our results reveled tht energy stress induces NBR expression t lest prtly through the LKB1 pthwy. NBR regultes mtorc1 signlling under energy stress To study the potentil function of NBR in mediting energy stress response, we generted 786-O cells ( kidney cncer cell line) nd MDA-MB-31 cells ( rest cncer cell line) with stle knockdown of NBR (Fig. ). We then nlysed whether knockdown of NBR ffected ny iochemicl signlling surrogte induced y energy stress, including ctivtion. As shown in Fig., glucose strvtion potently induced phosphoryltion of, or the sustrtes cetyl-coa croxylse (ACC) nd Rptor 18,8. Notly, NBR knockdown significntly ttenuted glucose-strvtioninduced phosphoryltion of, ACC nd Rptor. Accordingly, S6 nd S6K dephosphoryltion induced y glucose deprivtion ws significntly compromised in NBR knockdown cells compred with control short hirpin RNA (shrna)-infected cells (Fig. c). Finlly, NBR knockdown lso ttenuted DG- or A76966-tretmentinduced ctivtion nd mtorc1 inctivtion (Fig. d,e). Our results thus reveled tht NBR depletion ttenutes energy stress-induced ctivtion nd mtorc1 inctivtion, nd suggested feed-forwrd mechnism on NBR regultion, in which initilly promotes NBR expression in response to energy stress nd NBR in turn regultes ctivtion under energy stress (see Discussion). NBR regultes cell prolifertion, poptosis nd utophgy in response to energy stress functions s criticl metolic checkpoint; defective signlling leds to incresed cell prolifertion yet decresed utophgy under conditions of energy stress, leding to poptosis 1,. The forementioned dt prompted us to exmine the impct of NBR deficiency on cell prolifertion, poptosis nd utophgy in response to energy stress. Glucose strvtion mrkedly decresed S phse entry s mesured y BrdU incorportion, nd knockdown of NBR significntly ttenuted the reduction of S phse entry on glucose strvtion (Fig. 3 c). Thus, similr to cells with defective signlling 18, NBR-deficient cells continue cycling under energy stress. Although NBR depletion did not ffect poptosis under norml culture conditions, NBR deficiency induced more poptosis under glucose strvtion, s evidenced y oth Annexin V stining (Fig. 3d,e) nd cleved cspse-3 western lotting (Fig. 3f). In response to energy stress, ctivtes utophgy, cellulr dptive response to promote cell survivl under stress conditions,1. Accordingly, glucose-strvtion-induced GFP LC3 punct formtion, p6 degrdtion nd ULK1 phosphoryltion were significntly compromised in NBR-deficient cells (Fig. 3g,h nd Supplementry Fig. 3,), suggesting tht energy-stress-induced utophgy ws defective in NBR-deficient cells. Despite enhnced poptosis, the numer in NBR-deficient cells incresed under glucose-deprived conditions ecuse of the increse in cycling in NBR-deficient cells (Fig. 3i,j nd Supplementry Fig. 3c,d). Collectively, our results showed tht NBR deficiency leds to enhnced cell cycling yet decresed utophgy nd incresed poptosis under energy stress, which is in line with the phenotypes from cells with defective signlling, including -, LKB1-, TSC1- nd TSC-deficient cells or cells reconstituted with Rptor mutnt tht is non-phosphoryltle y (refs 15,18,19,9,3). NBR inhiits tumour development nd is downregulted in humn cncers Given the importnt functions of in the regultion of humn cncers, we next exmined the potentil roles of NBR in tumour development. NBR deficiency led to incresed nchorgeindependent growth, one of the hllmrks of cell trnsformtion, with more prominent effect under glucose-strvtion conditions (Fig.,). In vivo experiments using the xenogrft model showed tht NBR deficiency incresed tumour development (Fig. c). Further nlyses of the tumour smples y western lotting confirmed downregultion of nd upregultion of mtorc1 signlling in NBR-deficient tumours (Fig. d). Consistent with the experimentl results from rest nd renl cncer cell lines, survey of the RNA-seq dt cross different cncer types from the TCGA (The Cncer Genome Atls) dt sets reveled downregultion of NBR expression in rest (BRCA) nd renl (KIRC) cncer smples compred with pired norml tissue smples (Fig. e,f). Kpln Meier nlysis showed tht rest cncer ptients with NBR-low tumours hd significntly worse overll survivl thn those with NBR-high tumours (Fig. g). Together, our dt showed tht NBR deficiency promotes tumour development, nd NBR is downregulted in humn rest nd renl cncers, suggesting tht NBR my function s tumour suppressor in these cncers. Energy stress induces NBR interction with The forementioned iologicl dt prompted us to further study how NBR regultes function. Rel-time PCR nlyses of 3 16 Mcmilln Pulishers Limited. All rights reserved NATURE CELL BIOLOGY VOLUME 18 NUMBER APRIL 16

5 A RT I C L E S S phse (%) Apoptosis (%) Ctrl shrna NBR shrna1 P =. 1 3 NBR shrna P = Glucose (mm) Ctrl shrna NBR shrna1 NBR shrna P = P = Rtio of S phse (%) ( G/+G) 1 1 Glucose (mm) Glucose (mm) Apoptosis (%) Ctrl shrna 786-O c MDA-MB-31 P = P = shrna1 shrna d 786-O e f MDA-MB-31 Ctrl shrna P = NBR shrna1 NBR shrna P = Rtio of S phse (%) ( G/+G) MDA-MB O Ctrl NBR NBR Glucose shrna shrna1shrna (mm) M Cleved r (K) cspse-3 15 GAPDH Cleved cspse-3 GAPDH Ctrl shrna shrna1 shrna 15 g Cell with GFP LC3 punct (%) O Ctrl shrna P = 1. 1 NBR shrna1 NBR shrna h Cell with GFP LC3 punct (%) MDA-MB-31 Ctrl shrna NBR shrna1 P = NBR shrna i Reltive cell numers Glucose (mm) Glucose (mm) 786-O j MDA-MB-31 Ctrl shrna 1. Ctrl shrna NBR shrna1 1. NBR shrna1 NBR shrna NBR shrna P = P = Time (dys) Reltive cell numers 1 3 Time (dys) Figure 3 NBR regultes cell prolifertion, poptosis nd utophgy in response to energy stress. () Br grph showing the percentges of S phse (BrdU positive) cells in control-shrna- or NBR-shRNA-infected MDA-MB- 31 cells tht were cultured in or mm glucose-contining medium for h (men ± s.d., n=3 iologiclly independent extrcts, two-tiled pired Student s t-test). (,c) Br grph showing the glucose/+glucose rtio of S phse percentges in control-shrna- or NBR-shRNA-infected 786-O cells () or MDA-MB-31 cells (c) (men ± s.d., n = 3 iologiclly independent extrcts, two-tiled pired Student s t-test). (d f) Control-shRNA- or NBR- shrna-infected 786-O cells or MDA-MB-31 cells were cultured in medium with different concentrtions of glucose for h, nd then sujected to Annexin V/PI stining followed y FACS nlysis to mesure the percentges of Annexin V-positive/PI-negtive cells (d for 786-O cells, e for MDA-MB- 31 cells; men ± s.d., n = 3 iologiclly independent extrcts, two-tiled pired Student s t-test), or to western lotting nlysis to mesure cspse-3 clevge (f). (g,h) Br grph showing the percentges of cells with LC3 GFP punctte locliztion in control-shrna- or NBR-shRNA-infected 786-O cells (g) or MDA-MB-31 cells (h), which were trnsfected with GFP LC3 nd then cultured in or mm glucose-contining medium for 1 (for MDA-MB-31 cells) or 18 (for 786-O cells) h (men ± s.d., n = 5 fields per group, ech field ws ssessed from n independent experiment, twotiled pired Student s t-test). (i,j) 786-O (i) or MDA-MB-31 (j) cells infected with either control shrna or NBR shrna were cultured in glucosefree medium for different dys s indicted, nd then sujected to cell prolifertion nlysis (men ± s.d., n =3 iologiclly independent extrcts, two-tiled pired Student s t-test). Source dt for e,i,j cn e found in Supplementry Tle 1. Unprocessed originl scns of lots re shown in Supplementry Fig. 8. NATURE CELL BIOLOGY VOLUME 18 NUMBER APRIL Mcmilln Pulishers Limited. All rights reserved 35

6 A RT I C L E S c e NBR expression Numer of colonies Reltive tumour volume O MDA-MB-31 3 P = Control shrna NBR shrna1 NBR shrna Numer of colonies High glucose Low glucose High glucose Low glucose Ctrl shrna NBR shrna Time (weeks) 1 1 t: f t: Wilcox:.76 1 Wilcox: BRCA P = Reltive tumour volume NBR expression Tumour Norml Tumour Norml Time (weeks) Ctrl shrna NBR shrna KIRC d Control shrna NBR shrna1 NBR shrna P = 1. 1 Smple no. p- p-acc p-s6k p-s6 g Proility S Control shrna NBR shrna BRCA HR =.78 (.7.88) Log-rnk P = NBR high NBR low M r (K) Time (months) Figure NBR inhiits tumour development. (,) 786-O () or MDA-MB- 31 cells () infected with either control shrna or NBR shrna were seeded in soft gr contining high or low concentrtions of glucose s indicted. Br grph showing the men colony numers from the soft gr ssy (men ± s.d., n = 5 fields per group, ech field ws ssessed from n independent experiment, two-tiled pired Student s t-test). (c) Reltive tumour volumes of MDA-MB-31 xenogrft tumours infected with either control shrna or NBR shrna t different weeks (men ± s.e.m., n = 5 xenogrft tumours, : P <.5; : P <.1 two-tiled pired Student s t-test). (d) Protein lystes otined from xenogrft tumours infected with either control shrna or NBR shrna t the end point were sujected to western lotting nlysis s indicted. (e,f) The ox plot showing the expression pttern of NBR for ech pir of tumour nd norml smples in BRCA (e, n = 1 mtched pirs, Student s t-test nd Wilcoxon test) nd KIRC (f, n = 65 mtched pirs, Student s t-test nd Wilcoxon test). The oxes show the medin ±1 qurtile, with whiskers extending to the most extreme dt point within 1.5 interqurtile rnge from the ox oundries. (g) Kpln Meier plots of rest cncer ptients strtified y the expression levels of NBR (n high = 1,767, n low = 1,787, log-rnk test). Unprocessed originl scns of lots re shown in Supplementry Fig. 8. frctionted nucler nd cytoplsmic RNA reveled tht NBR loclized in oth the nucleus nd the cytoplsm (Fig. 5). As expected, α showed predominnt locliztion in the cytoplsm (Fig. 5). exists s heterotrimeric complex tht consists of ctlytic α suunit nd two regultory β nd γ suunits 31. We thus exmined whether NBR cn interct with ny of the suunits of y RNA-pulldown ssy using in vitro-synthesized iotinylted NBR. Such nlysis reveled tht NBR intercted with overexpressed α under glucose-strvtion conditions, with miniml inding with overexpressed β or γ suunit (Fig. 5c). The RNA-pulldown ssy lso reveled tht glucose strvtion significntly incresed the interction of NBR with endogenous α (Fig. 5d). As α, β nd γ suunits form very stle complex t the endogenous level, we lso oserved glucose-strvtion-induced inding etween NBR nd endogenous β nd γ suunits (Fig. 5d), proly medited y NBR interction with the endogenous α suunit. In vitro inding ssy using purified α nd in vitro-synthesized iotinylted NBR confirmed the direct inding etween NBR nd α (Fig. 5e). There exist t lest three splicing isoforms of the NBR gene (nmed s NBR no. 1, no. nd no. 3; see Supplementry Fig. 1). In the RNA-pulldown experiments descried ove, we used the NBR no. 1 splicing isoform. The RNA-pulldown experiments showed tht the NBR no. nd no. 3 splicing isoforms lso intercted with α on glucose strvtion (Fig. 5f). Finlly, Mcmilln Pulishers Limited. All rights reserved NATURE CELL BIOLOGY VOLUME 18 NUMBER APRIL 16

7 A RT I C L E S c Reltive expression NBR 1.5 GAPDH 1.5 U1 P = 1. 1 P = Reltive expression 1..5 Totl Nul Cyt Totl Nul Cyt Totl Nul Cyt Glucose (mm) Flg Flg α Input AS NBR no. 1 pulldown S NBR no. 1 pulldown Reltive expression Input AS NBR no. 1 pulldown S NBR no. 1 pulldown Flg β 1..5 Flg γ Input AS NBR no. 1 pulldown S NBR no. 1 pulldown d HSP9 PARP α Glucose (mm) p- α β γ1 Totl Nul Cyt M r (K) 1 1 Input AS NBR no. 1 pulldown S NBR no. 1 pulldown M r (K) e Glucose (mm) α AS NBR no. pulldown S NBR no. pulldown AS NBR no. 3 pulldown S NBR no. 3 pulldown f α Input AS NBR no. 1 pulldown S NBR no. 1 pulldown g Reltive NBR level Input lgg P = P =.6 1 Glucose (mm) h WT 17A NT α T17 Kinse domin A17 T17 NBR inding Flg IB: Flg WT α 17A NT CT WT 17A NT CT β α β M r (K) 75 CT Input NBR no. 1 pulldown Figure 5 Energy stress induces NBR interction with. (,) Nucler nd cytoplsmic frctions of 786-O cells were sujected to either reltime PCR (, men ± s.d., n = 3 iologiclly independent extrcts, twotiled pired Student s t-test) or western lotting nlysis (). (c) In vitrosynthesized iotinylted sense (S) or ntisense (AS) NBR no. 1 ws incuted with protein lystes from HEK93T cells trnsfected with vrious vectors s indicted. Precipittion rections were conducted using streptvidin eds nd then sujected to western lotting. (d,e) In vitrosynthesized iotinylted sense (S) NBR or ntisense (AS) NBR with different splicing isoforms ws incuted with protein lystes from 786-O cells tht hd een cultured in or mm glucose-contining medium for h. Precipittion rections were conducted using streptvidin eds nd then sujected to western lotting. (f) In vitro-synthesized iotinylted sense (S) or ntisense (AS) NBR no. 1 ws incuted with purified humn α protein. Precipittion rections were conducted using streptvidin eds nd then sujected to western lotting. (g) 786-O cells were cultured in or mm glucose-contining medium for h. Protein lystes were prepred nd immunoprecipitted with α ntiody or IgG. The RNA levels of NBR in immunoprecipittes or cell lystes (input) were mesured y reltime PCR (men ± s.d., n = 3 iologiclly independent extrcts, two-tiled pired Student s t-test). (h) In vitro-synthesized iotinylted NBR no. 1 ws incuted with protein lystes from HEK93T cells trnsfected with vrious vectors nd sujected to glucose strvtion. Precipittion rections were conducted using streptvidin eds nd then sujected to western lotting. Source dt for,g cn e found in Supplementry Tle 1. Unprocessed originl scns of lots re shown in Supplementry Fig. 8. RNA immunoprecipittion ssy (using primers tht cn detect ll three NBR splicing isoforms) reveled n enrichment of NBR in the precipittes of α compred with the IgG control, nd glucose strvtion sustntilly incresed the enrichment of NBR in α precipittes (note tht glucose strvtion resulted in much higher fold increse of the NBR level in α precipittes compred with the NBR input level) (Fig. 5g). In the experiment to mp the region(s) of α tht medites interction with NBR, we showed tht the kinse-domincontining mino-terminl region, ut not the croxy-terminl NATURE CELL BIOLOGY VOLUME 18 NUMBER APRIL Mcmilln Pulishers Limited. All rights reserved 37

8 A RT I C L E S region of α, intercted with NBR (Fig. 5h). Muttion of Thr17 to lnine in α did not ffect α interction with NBR (Fig. 5h), indicting tht phosphoryltion t Thr17 is not required for NBR interction. Together, our dt reveled tht glucose strvtion not only induces NBR expression, ut lso enhnces NBR interction with, which is possily medited y NBR interction with the kinse domin of α. NBR promotes kinse ctivity Next we studied the underlying mechnisms y which NBR regultes function. To this end, we first exmined whether overexpression of NBR exerts ny iologicl effect in cells. Our experiments reveled tht overexpression of ny of the three NBR splicing isoforms resulted in ctivtion, mtorc1 inctivtion (Fig. 6,), nd decresed cell prolifertion without ffecting poptosis under norml culture conditions (Fig. 6c). All three splicing isoforms of NBR shre the sme first two exons locted t the 5 end of NBR with distinctive exons locted towrds the 3 end (Supplementry Fig. 1). Our dt thus indicte tht the common exons in ll NBR splicing isoforms might e importnt in mediting NBR interction with. Consistent with this, our inding mpping experiments reveled tht the first exon shred y ll three NBR splicing isoforms is oth required nd sufficient to medite NBR interction with α (Fig. 6d). Furthermore, overexpression of the T1 frgment of NBR no. 1, which lcks the first exon (with 159 se pirs (p) out of 1,5 p full-length NBR no. 1) nd thus is incple of intercting with, did not ffect nd mtorc1 ctivtion sttus or cell prolifertion, wheres in the prllel experiments, overexpression of full-length NBR no. 1 exerted the expected effects on signlling (Fig. 6e,f). It seems tht overexpression of the first exon lone (T frgment of NBR no. 1) ws not sufficient to ctivte (Supplementry Fig. ), suggesting tht other regions in NBR my e lso importnt for NBR function in the regultion of. Together, our results showed tht deletion of the first exon of NBR olishes its interction with nd regultion of ctivtion, suggesting tht NBR regultion of ctivtion nd downstrem cellulr processes is proly medited through NBR interction with. As LKB1 functions s the min upstrem kinse of in response to energy stress 13 15, we exmined whether NBR regultes LKB1 interction with. Our results showed tht NBR overexpression or knockdown did not ffect LKB1 interction under either sl or glucose-strvtion conditions (Supplementry Fig.,c). In ddition, we found tht overexpression of NBR in LKB1-deficient HeL cells could still promote ctivtion, nd co-expression of NBR nd LKB1 in HeL cells led to synergistic increse of ctivtion (Supplementry Fig. d). Together, our dt suggest tht NBR does not regulte LKB1 interction nd it is likely tht NBR opertes in prllel to LKB1 to regulte ctivtion. Our dt showing tht NBR intercts with the kinse domin of α (Fig. 5h) prompted the hypothesis tht NBR my directly regulte the kinse ctivity of the complex. Our dt showed tht cterilly purified GST ACC (mino cids 1 13) could e redily phosphorylted y the complex precipitted from cell lystes of HEK93T cells co-trnsfected with α/β/γ constructs (Fig. 6g). Wheres in vitro-synthesized NBR lone did not led to ACC phosphoryltion, the ddition of NBR (ut not the T1 frgment of NBR, the non-inding mutnt) to the complex significntly incresed ACC phosphoryltion y (Fig. 6g). The in vitro kinse ssy using purified α/β/γ complex nd SAMS peptide s the sustrte further confirmed tht NBR promoted in vitro kinse ctivity (Fig. 6h). Together, our dt suggest tht NBR functions to promote kinse ctivity possily through its interction with the kinse domin. The functionl effects of NBR re prtilly medited y We next sought to determine the extent to which the functionl effects of NBR re medited y NBR regultion of ctivtion. We first exmined whether overexpression of NBR still exerted its functionl effects in α knockdown cells. Such nlyses reveled tht, lthough overexpression of NBR incresed ACC phosphoryltion, decresed S6 phosphoryltion, nd suppressed cell prolifertion in control (Ctrl)-siRNA-trnsfected cells, such effects were ttenuted in α knockdown ( sirna) cells (Fig. 7,). As complementry pproch, we lso exmined whether restortion of constitutively ctive (CA) (mino cids 1 31 of α1) would rescue ny of the defects oserved in NBR-deficient cells. Our dt reveled tht overexpression of CA in NBR knockdown cells restored ACC or S6 phosphoryltion under glucosestrvtion conditions s expected (Fig. 7c), nd correspondingly, significntly rescued cell prolifertion, poptosis, nd nchorgeindependent growth under glucose-strvtion conditions in NBR- deficient cells (Fig. 7d g). Importntly, restortion of CA in the NBR-deficient ckground significntly ttenuted the enhnced xenogrft tumour development cused y NBR deficiency (Fig. 7h). Tken together, our dt strongly suggested tht the functionl effects of NBR re t lest prtilly dependent on. DISCUSSION exists s heterotrimeric complex comprising of ctlytic α suunit nd two regultory β nd γ suunits, in which the γ suunit directly inds to AMP in response to energy stress 31. It hs een proposed tht AMP ctivtes through t lest three mechnisms: AMP inding to cuses llosteric ctivtion of, nd leds to conformtionl chnge of the complex tht promotes Thr17 phosphoryltion in the α suunit y LKB1 nd inhiits Thr17 dephosphoryltion y protein phosphtses 31. Our study revels tht lincrna NBR regultion of represents nother importnt regultory mechnism to control ctivtion in response to energy stress. Here we propose feed-forwrd model on NBR regultion. Specificlly, energystress-induced initil ctivtion does not require NBR. Activted then upregultes NBR expression in response to energy stress. NBR in turn intercts with nd promotes kinse ctivity under energy stress, forming feed-forwrd loop to potentite ctivtion during chronic energy stress conditions (Supplementry Fig. 5). NBR deficiency leds to inctivtion during long periods of energy stress, which promotes mtorc1 ctivtion, cell prolifertion nd tumour development (Supplementry Fig. 5). As trnscription regultion in generl tkes Mcmilln Pulishers Limited. All rights reserved NATURE CELL BIOLOGY VOLUME 18 NUMBER APRIL 16

9 A RT I C L E S p- p-acc ACC NBR NBR EV No. 1 No. No. 3 EV No. 1 No. No. 3 c Reltive cell numers EV No. 1 No. No. 3 UMRC P = d p-s6k S6K HEK93T NBR no. 1 Exon 1 3 α RNA AS S T1 T3 Input UMRC AS S T T5 T6 Input AS S (FL) T1 T3 T T5 T6 inding f g Reltive cell numers 1 3 Time (dys) 8 EV No. 1 FL 6 No. 1 T1 1 3 Time (dys) p-acc I GST ACC 1 13 NBR no. 1 FL + NBR no. 1 FL + NBR no. 1 T1 P = e p- p-s6 S6 NBR no. 1 NBR no. 1 EV FL T1 EV FL T1 HEK93T UMRC M r (K) h Kinse ctivity (percentge of control) Control P =. 1 P = 3. 1 P =.6 1 S NBR (μg) Compound C (μm) A76996 (μm) AS NBR T AMP (μm) Figure 6 NBR promotes kinse ctivity. (,) Protein lystes were prepred from HEK93T () or UMRC cells () with overexpression of EV or NBR expression vectors, nd nlysed y western lotting. (c) UMRC cells stly expressing EV or NBR expression vectors were cultured in mm glucose-contining medium for different dys s indicted, nd then sujected to cell prolifertion nlysis (men ± s.d., n = 3 iologiclly independent extrcts, two-tiled pired Student s t-test). (d) Top: Schemtic digrm showing different trunction mutnts of NBR no. 1 nd the summry of their inding cpilities to α. Bottom: In vitro-synthesized iotinylted sense (S), ntisense (AS), or different trunction (T) mutnts of NBR no. 1 were incuted with protein lystes from 786-O cells tht hd een cultured in glucose-free medium for h. Precipittion rections were conducted using streptvidin eds nd then sujected to western lotting. (e) Protein lystes were prepred from HEK93T or UMRC cells with overexpression of EV, NBR no. 1 full length (FL), or T1 mutnt expression vectors, nd nlysed y western lotting. (f) UMRC cells stly expressing EV, NBR no. 1 FL, or T1 mutnt expression vectors were cultured in mm glucose-contining medium for different numers of dys s indicted, nd then sujected to cell prolifertion nlysis (men ± s.d., n =3 iologiclly independent extrcts, two-tiled pired Student s t-test). (g) complex precipitted from HEK93T cells ws sujected to the kinse ssy in the presence of ATP, in vitro-synthesized RNAs nd GST ACC 1 13 mino cid fusion proteins s indicted. The kinse ctivity of ws mesured y phosphoryltion of ACC t the Ser79 site. (h) In vitro-purified ctive humn complex ws sujected to in vitro kinse ssys in the presence of ATP, SAMS peptide nd in vitro-synthesized iotinylted sense (S)/ntisense (AS)/T1 mutnt (T1) NBR no. 1 or severl chemicl compounds (compound C, A76966, AMP) s indicted (see Methods for detils). The kinse ctivity ws mesured y the luminescence with plte-reding illuminometer (men ± s.d., n = 3 iologiclly independent extrcts, two-tiled pired Student s t-test). Source dt for c,f,h cn e found in Supplementry Tle 1. Unprocessed originl scns of lots re shown in Supplementry Fig. 8. NATURE CELL BIOLOGY VOLUME 18 NUMBER APRIL Mcmilln Pulishers Limited. All rights reserved 39

10 A RT I C L E S p- c p-acc Numer of colonies ACC EV NBR no. 1 High glucose EV NBR no. 1 EV CA Ctrl NBR Ctrl NBR shrna shrna shrna shrna +G G +G G +G G +G G p-acc/acc p-s6 S6 p-s6/s V5 ( CA) e g p-acc Apoptosis (%) ACC p-s6 S6 Ctrl sirna sirna1 Ctrl sirna sirna1 Ctrl sirna sirna Ctrl sirna sirna P = P = mm Glu mm Glu Ctrl shrna + EV NBR shrna + EV Ctrl shrna + CA NBR shrna + CA Ctrl shrna + EV Ctrl shrna + CA NBR shrna + EV NBR shrna + CA Low glucose M r (K) M r (K) P = P = d f h Reltive cell numers Reltive cell numers Reltive cell numers Cleved PARP Tuulin Reltive tumour volume 1 EV+ Ctrl sirna EV+ sirna1 NBR no. 1 + Ctrl 8 NBR no. 1 + sirna Time (dys) EV+ Ctrl sirna EV+ sirna NBR no. 1 + Ctrl sirna NBR no. 1 + sirna 1 3 Time (dys) 1 3 Time (dys) Ctrl shrna EV NBR shrna Ctrl shrna + EV Ctrl shrna + CA NBR shrna + EV NBR shrna + CA Ctrl shrna CA NBR shrna +G G +G G +G G +G G Ctrl shrna + EV NBR shrna + CA NBR shrna + EV 1 3 Time (weeks) P =.5 1 P =.7 1 P =. 1 P = P = M r (K) 1 Figure 7 The functionl effects of NBR re prtilly medited y. (,) UMRC cells stly expressing EV or NBR expression vectors were trnsfected with sirna ( sirna1 or sirna) or control (Ctrl) sirna. Protein lystes were prepred nd nlysed y western lotting (), or cells were cultured in mm glucose-contining medium for different numers of dys s indicted, nd then sujected to cell prolifertion nlysis () (men ± s.d., n=3 iologiclly independent extrcts, two-tiled pired Student s t-test). (c g) MDA-MB-31 cells with stle expression of control (Ctrl) shrna or NBR shrna were infected with empty vector (EV) or constitutively ctive ( CA). These cells were cultured in or mm glucose-contining medium for h, nd protein lystes were prepred nd nlysed y western lotting (c); the cells were cultured in mm glucose-contining medium for different numers of dys s indicted, nd then sujected to crystl violet stining to mesure cell numer (d) (men ± s.d., n = 3 iologiclly independent extrcts, two-tiled pired Student s t-test); the cells were cultured in or mm glucose-contining medium for h, nd then sujected to Annexin V/PI stining followed y FACS nlysis to mesure the percentges of Annexin V-positive/PI-negtive cells (e) (men ± s.d., n = 5 fields per group, ech field ws ssessed from n independent experiment, two-tiled pired Student s t-test), or to western lotting nlysis to mesure PARP clevge (f); the cells were seeded in soft gr contining high or low concentrtions of glucose s indicted. Br grph showing the men colony numers from the soft gr ssy (g) (men ± s.d., n = 5 fields per group, ech field ws ssessed from n independent experiment, two-tiled pired Student s t-test). (h) Reltive tumour volumes of MDA-MB-31 xenogrft tumours of different genotypes t different weeks (men ± s.d., n = 5 xenogrft tumours, : P <.5, : P <.1, two-tiled pired Student s t-test). Source dt for,d,e cn e found in Supplementry Tle 1. Unprocessed originl scns of lots re shown in Supplementry Fig Mcmilln Pulishers Limited. All rights reserved NATURE CELL BIOLOGY VOLUME 18 NUMBER APRIL 16

11 A RT I C L E S longer time thn llosteric regultion nd phosphoryltion events, we reson tht cells my hve evolved this lincrna-involved regultory mechnism to mintin ctivtion during long periods of energy stress nd to help cells dpt etter to chronic stress conditions. In support of this model, our time course experiments reveled tht NBR deficiency compromised ctivtion t lter, ut not erlier, time points on glucose strvtion (Supplementry Fig. 6). (Note tht ll of the energy stress experiments shown in our studies used 1 h or longer tretment time points.) This mirrors well with the kinetics of NBR expression induction on glucose strvtion (Supplementry Fig. 6). As glucose strvtion lso significntly promotes NBR inding to (Fig. 5), this presumly further mplifies the effect of NBR to promote ctivtion. The NBR gene ws originlly identified s gene tht is locted ner to the rest-cncer-ssocited gene BRCA1. Both genes lie hed to hed with ech other on humn chromosome 17, nd the physicl distnce etween the trnscription strt sites of the two genes is only 18 p (Supplementry Fig. 1) 7. Given the frequent muttion/deletion rtes of BRCA1 in humn rest nd ovrin cncers nd the close proximity of the NBR gene to the BRCA1 gene, it ws initilly postulted tht NBR should e co-deleted/mutted with BRCA1 in certin cncers (for exmple, see ref. 3), nd NBR my lso ply role in tumour suppression. However, lter it ecme cler tht NBR does not seem to encode protein, nd it ws proposed tht NBR simply is junk gene 33. Since then, its potentil function in tumour iology hs remined unknown. In this study, we identified NBR s lincrna induced y energy stress, nd showed tht NBR indeed functions to inhiit tumour development, t lest in prt through its regultion of ctivtion. It is of note tht NBR overexpression in -deficient cells cn still exert moderte cell prolifertion suppressive effect (Fig. 7), suggesting tht NBR my hve other -independent function(s) to regulte cell prolifertion. Identifiction nd chrcteriztion of other NBR- inding proteins or RNAs will further clrify its function. The most populr model proposed for lncrna function proly is the one wherey lncrnas regulte gene expression, either in cis or in trns, through recruiting other chromtin-modifiction complexes or trnscription fctors to specific loci 3,35. This rises the possiility tht NBR my regulte the trnscription of the BRCA1 gene, which resides right next to the NBR gene. However, our dt showed tht BRCA1 expression ws not ffected y either glucose strvtion or NBR knockdown (Supplementry Fig. 7). We should mention tht, lthough initilly it ws proposed tht lincrnas minly function to regulte neighouring gene trnscription, other studies hve shown tht mny lincrnas do not exert such function 1. Whether NBR regultes ny other gene trnscription wits further investigtion. METHODS Methods nd ny ssocited references re ville in the online version of the pper. Note: Supplementry Informtion is ville in the online version of the pper ACKNOWLEDGEMENTS We thnk ll memers of the Gn lortory for their dvice nd technicl ssistnce. This reserch hs een supported y grnts from MD Anderson Cncer Center, US Deprtment of Defense (TS939), Cncer Prevention & Reserch Institute of Texs (RP13), Ntionl Institutes of Helth (CA nd CA1937), Ellison Medicl Foundtion (AG-NS ), nd Grielle s Angel Foundtion for Cncer Reserch (to B.G.). B.G. is Kimmel Scholr nd n Ellison Medicl Foundtion New Scholr. H.Ling is supported y the Ntionl Institutes of Helth (CA13883, CA17586); the R. Lee Clrk Fellow Awrd from The Jenne F. Shely Scholrship Fund; grnt from the Cncer Prevention nd Reserch Institute of Texs (RP16); nd the Mry K. Chpmn Foundtion nd the Lorrine Dell Progrm in Bioinformtics for Personliztion of Cncer Medicine. L.H. is supported y Cncer Prevention & Reserch Institute of Texs (RR185). H.-K.L. is supported y the Ntionl Institutes of Helth (CA18 nd CA193813). B.G., J.C., J.W. nd H.Ling re memers of the MD Anderson Cncer Center, nd re supported y the Ntionl Institutes of Helth Core Grnt CA1667. AUTHOR CONTRIBUTIONS Z.-D.X. initited the project nd identified NBR s n energy-stress-induced lncrna. X.L. performed most of the experiments with ssistnce from Z.-D.X., H.Lee, nd L.Z. J.Z. nd J.W. nlysed RNA-seq dt set. L.H. nd H.Ling conducted computtionl nlysis on NBR expression nd sttus in humn cncers. W.W., J.C., S.-W.L. nd H.-K.L. provided regents. B.G. supervised the study. X.L., Z.-D.X. nd B.G. designed the experiments nd wrote the mnuscript. All uthors commented on the mnuscript. COMPETING FINANCIAL INTERESTS The uthors declre no competing finncil interests. Pulished online t Reprints nd permissions informtion is ville online t 1. Ulitsky, I. & Brtel, D. P. lincrnas: genomics, evolution, nd mechnisms. Cell 15, 6 6 (13).. EP Consortium, An integrted encyclopedi of DNA elements in the humn genome. Nture 89, 57 7 (1). 3. Btist, P. J. & Chng, H. Y. Long noncoding RNAs: cellulr ddress codes in development nd disese. Cell 15, (13).. Gupt, R. A. et l. Long non-coding RNA HOTAIR reprogrms chromtin stte to promote cncer metstsis. Nture 6, (1). 5. Hurte, M. et l. A lrge intergenic noncoding RNA induced y p53 medites glol gene repression in the p53 response. Cell 1, 9 19 (1). 6. Prensner, J. R. et l. Trnscriptome sequencing cross prostte cncer cohort identifies PCAT-1, n unnnotted lincrna implicted in disese progression. Nt. Biotechnol. 9, 7 79 (11). 7. Engreitz, J. M. et l. The Xist lncrna exploits three-dimensionl genome rchitecture to spred cross the X chromosome. Science 31, (13). 8. Rinn, J. L. et l. Functionl demrction of ctive nd silent chromtin domins in humn HOX loci y noncoding RNAs. Cell 19, (7). 9. Wng, K. C. et l. A long noncoding RNA mintins ctive chromtin to coordinte homeotic gene expression. Nture 7, 1 1 (11). 1. Guttmn, M. & Rinn, J. L. Modulr regultory principles of lrge non-coding RNAs. Nture 8, (1). 11. vn Heesch, S. et l. Extensive locliztion of long noncoding RNAs to the cytosol nd mono- nd polyriosoml complexes. Genome Biol. 15, R6 (1). 1. Hrdie, D. G., Ross, F. A. & Hwley, S. A. : nutrient nd energy sensor tht mintins energy homeostsis. Nt. Rev. Mol. Cell Biol. 13, 1 6 (1). 13. Hwley, S. A. et l. Complexes etween the LKB1 tumor suppressor, STRAD α/β nd MO α/β re upstrem kinses in the AMP-ctivted protein kinse cscde. J. Biol., 8 (3). 1. Woods, A. et l. LKB1 is the upstrem kinse in the AMP-ctivted protein kinse cscde. Curr. Biol. 13, 8 (3). 15. Shw, R. J. et l. The tumor suppressor LKB1 kinse directly ctivtes AMP-ctivted kinse nd regultes poptosis in response to energy stress. Proc. Ntl Acd. Sci. USA 11, (). 16. Hrdie, D. G., Schffer, B. E. & Brunet, A. : n energy-sensing pthwy with multiple inputs nd outputs. Trends Cell Biol. 6, 19 1 (15). 17. Lplnte, M. & Stini, D. M. mtor signling in growth control nd disese. Cell 19, 7 93 (1). 18. Gwinn, D. M. et l. phosphoryltion of rptor medites metolic checkpoint. Mol. Cell 3, 1 6 (8). 19. Inoki, K., Zhu, T. & Gun, K. L. TSC medites cellulr energy response to control cell growth nd survivl. Cell 115, (3).. Egn, D. F. et l. Phosphoryltion of ULK1 (hatg1) y AMP-ctivted protein kinse connects energy sensing to mitophgy. Science 331, (11). 1. Kim, J., Kundu, M., Viollet, B. & Gun, K. L. nd mtor regulte utophgy through direct phosphoryltion of Ulk1. Nt. Cell Biol. 13, (11).. Shckelford, D. B. & Shw, R. J. The LKB1- pthwy: metolism nd growth control in tumour suppression. Nt. Rev. Cncer 9, (9). 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12 A RT I C L E S 3. Hung, J. & Mnning, B. D. The TSC1-TSC complex: moleculr switchord controlling cell growth. Biochem. J. 1, (8).. Hezel, A. F. & Brdeesy, N. LKB1; linking cell structure nd tumor suppression. Oncogene 7, (8).. Alessi, D. R., Skmoto, K. & Byscs, J. R. LKB1-dependent signling pthwys. Annu. Rev. Biochem. 75, (6). 6. Fuert, B. et l. is negtive regultor of the Wrurg effect nd suppresses tumor growth in vivo. Cell Met. 17, (13). 7. Xu, C. F. et l. Isoltion nd chrcteristion of the NBR gene which lies hed to hed with the humn BRCA1 gene. Hum. Mol. Genet. 6, (1997). 8. Sim, A. T. & Hrdie, D. G. The low ctivity of cetyl-coa croxylse in sl nd glucgon-stimulted heptocytes is due to phosphoryltion y the AMP-ctivted protein kinse nd not cyclic AMP-dependent protein kinse. FEBS Lett. 33, 9 98 (1988). 9. Bungrd, D. et l. Signling kinse ctivtes stress-promoted trnscription vi histone HB phosphoryltion. Science 39, 11 1 (1). 3. Corrdetti, M. N., Inoki, K., Brdeesy, N., DePinho, R. A. & Gun, K. L. Regultion of the TSC pthwy y LKB1: evidence of moleculr link etween tuerous sclerosis complex nd Peutz-Jeghers syndrome. Genes Dev. 18, (). 31. Hrdie, D. G. -sensing energy while tlking to other signling pthwys. Cell Met., (1). 3. Gd, S. et l. Chrcteristion of 161 k deletion extending from the NBR1 to the BRCA1 genes in French rest-ovrin cncer fmily. Hum. Mutt. 1, 65 (3). 33. Jin, H. et l. Structurl evolution of the BRCA1 genomic region in primtes. Genomics 8, (). 3. Pndey, R. R. et l. Kcnq1ot1 ntisense noncoding RNA medites linegespecific trnscriptionl silencing through chromtin-level regultion. Mol. Cell 3, 3 6 (8). 35. Feng, J. et l. The Evf- noncoding RNA is trnscried from the Dlx-5/6 ultrconserved region nd functions s Dlx- trnscriptionl coctivtor. Genes Dev., (6). 16 Mcmilln Pulishers Limited. All rights reserved NATURE CELL BIOLOGY VOLUME 18 NUMBER APRIL 16

13 DOI: 1.138/nc338 M E T H O D S METHODS Cell culture studies. Humn kidney cncer cell lines, humn rest cncer cell lines, humn prostte cncer cell lines, humn emryonic kidney 93 cells used in this study were mostly otined from Americn Type Culture Collection (ATCC). All of the cell lines were free of mycoplsm contmintion (tested y the vendors using the MycoAlert kit from Lonz). No cell lines used in this study re found in the dtse of commonly misidentified cell lines (ICLAC nd NCBI Biosmple) sed on short tndem repet (STR) profiling performed y vendors. HeL or A59 cells with expression of empty vector or Lk1 expression vectors were descried in ref. 36. sirna nd plsmid trnsfections were performed using Lipofectmine 3 (Life Technologies). Lentiviruses or retroviruses were produced in HEK93T cells with pcking mix (VirPower Lentivirl Expression System, Invitrogen) nd used to infect trget cells s per the mnufcturer s instructions. For glucose-strvtion experiments, cells were cultured in DMEM with different concentrtions of glucose (, 1, or mm) + 1% dilysed FBS. To mesure poptosis, the cells were stined y the Annexin V kit s per the mnufcturer s instructions (BD Bioscience) 37. Briefly, treted cells were wshed with PBS twice nd then cells were resuspended in 1 µl of 1 inding uffer. FITC-lelled Annexin V nd propidium iodide were dded to smples nd incuted in the drk for 15 min t room temperture. Susequently, cells were sujected to FACS nlysis. Cell cycle nlysis ws crried out s previously reported using the FITC BRDU Flow Kit (BD Bioscience) 38,39. Cell growth nd soft gr ssys were conducted s descried in our previous pulictions,1. Briefly, for cell growth ssy, cells were plted in -well pltes nd growth ws determined y crystl violet stining. Cells were stined with.1% crystl violet (Sigm) solution for 15 min t room temperture. Stined crystl violet ws then extrcted with 1% cetic cid nd the intensity of the colour ws mesured y photospectrometry t OD 595. To ssess nchorge-independent growth, 1, cells per well in.% grose on top of ottom lyer of.7% grose were seeded in triplicte wells of 6-well pltes. On the formtion of colonies, soft gr pltes were stined with iodonitrotetrzolium chloride (Sigm) nd the colonies were counted mnully. Constructs nd regents. shrnas trgeting humn NBR (NM_ s1c1, NM_ s1c1) were purchsed from Sigm (note tht these two shrnas trget splicing isoforms no. 1 nd no. 3 of NBR, nd cn still chieve good knockdown efficiency when mesured y rel-time PCR primer set designed to detect ll three splicing isoforms of NBR). sirnas trgeting α were purchsed from Origene (SR3371, SR337). All three splicing isoforms of NBR were otined from Thermo Fisher Scientific (MGC humn NBR sequenceverified cdnas, clone ID: 6595, 33997, 86858) nd then were sucloned into Lentivirl vector plvx (Clontech). α, β nd γ entry plsmids were otined from the Humn ORFeome V5.1 lirry. The entry clones were susequently recomined into gtewy-comptile destintion expression vectors with Flg tg through LR Gtewy Technology (Invitrogen). cdna corresponding to mino cids 1 31 of α1 ws cloned into entry vector, nd ws susequently recomined into gtewy-comptile destintion expression vectors with V5 tg through LR Gtewy Technology (Invitrogen). Active humn α protein nd ctive humn α1+β1+γ1 protein were purchsed from Acm (7983, 16916). -Deoxy-D-glucose nd compound C were purchsed from Sigm (D613, P599). A ws purchsed from LC lortories (A-183). Quntittive rel-time PCR nd RNA immunoprecipittion (RIP) ssy. Totl RNA ws extrcted from cells using RNesy (Qigen) nd first-strnd cdna ws prepred with the High Cpcity cdna Reverse Trnscription Kit (Applied Biosystems, ABI). Rel-time PCR ws performed using the QuntiTect SYBR Green PCR kit (Qigen) or TqMn Universl PCR Mster Mix (ABI), nd ws run on the Strtgene MX3P. For quntifiction of gene expression, the Ct method ws used. GAPDH expression ws used for normliztion. RIP ssy ws performed with the Mgn RIP RNA-Binding Protein Immunoprecipittion Kit (Millipore). Briefly, cells were lysed in RIP lysis uffer. Then the lystes were immunoprecipitted with ntiody or IgG long with protein mgnetic eds. After proteinse K digestion, the RNAs pulled down with proteins were purified y phenol chloroform extrction nd precipitted in ethnol. The RNAs were then resuspended in RNAse-free wter nd cdna ws synthesized nd sujected to rel-time PCR to detect NBR or GAPDH (internl control) trnscripts. The RNA level ws normlized with input (1%). RNA-pulldown ssys. Biotin-lelled RNAs were synthesized y the Scientific TrnscriptAid T7 High Yield Trnscription Kit (Thermo). PCR primers with T7 promoters were used to mplify DNA templtes for RNA synthesis, RNA trnscried in vitro with iotin RNA lelling mix nd T7 RNA polymerse, treted with RNse-free DNse I (Roche), nd purified with the RNesy Mini Kit (Qigen). Cells lystes or purified proteins were incuted with iotin-lelled RNAs overnight. The proteins ssocited with iotin-lelled RNAs were then pulled down with streptvidin mgnetic eds (Thermo) fter 1-h incution. The proteins ws then wshed nd used for western lot nlysis. Western lot nlysis. Tissues were lysed with RIPA uffer ( mm Tris ph 7.5, 1 mm NCl, 1% Nonidet P-,.5% sodium deoxycholte, 1 mm EDTA,.1% SDS) contining complete mini protese inhiitors (Roche) nd phosphtse inhiitor cocktil (Cliochem). Cultured cells were lysed with NP uffer (1 mm sodium chloride, 1.% NP-, mm Tris, ph 8.) contining complete mini protese inhiitors (Roche) nd phosphtse inhiitor cocktil (Cliochem). Western lots were otined using to µg of lyste protein. The following ntiodies were used in this study: nti-flag tg (M) mouse monoclonl ntiody (Sigm-Aldrich, F3165, 1:5, dilution), monoclonl ntivinculin ntiody (Sigm-Aldrich, V5, 1:5, dilution), phospho-cetyl-coa croxylse (Ser79) ntiody (Cell Signling Technology, 3661S, 1:1, dilution), cetyl-coa croxylse ntiody (Cell Signling Technology, 366S, 1:1, dilution), phospho-p7 S6 kinse (Thr389) ntiody (Cell Signling Technology, 9S, 1:1, dilution), phospho-α (Thr17) (H9) rit monoclonl ntiody (Cell Signling Technology, 35S, 1:1, dilution), α (D63G) rit monoclonl ntiody (Cell Signling Technology, 583S, 1:1, dilution), α (F6) mouse monoclonl ntiody (Cell Signling Technology, 793S, 1:1, dilution), β1/ (57C1) rit monoclonl ntiody (Cell Signling Technology, 1S, 1:1, dilution), γ1 ntiody (Cell Signling Technology, 187S, 1:1, dilution), phospho-rptor (Ser79) ntiody (Cell Signling Technology, 83S, 1:1, dilution), Rptor (C1) rit monoclonl ntiody (Cell Signling Technology, 8S, 1:1, dilution), phospho-s6 riosoml protein (Ser/) (D68F8), XP rit monoclonl ntiody (Cell Signling Technology, 536S, 1:5, dilution), S6 riosoml protein (5G1) rit monoclonl ntiody (Cell Signling Technology, 17S, 1:5, dilution), ULK1 (D8H5) rit monoclonl ntiody (Cell Signling Technology, 85 S, 1:1, dilution), phospho-ulk1 (Ser555) (D1H) rit monoclonl ntiody (Cell Signling Technology, 5869S, 1:1, dilution), phospho-ulk1 (Ser757) ntiody (Cell Signling Technology, 6888S, 1:1, dilution), cleved PARP (Asp1) ntiody (Humn Specific) (Cell Signling Technology, 951S, 1:, dilution), PARP ntiody (Cell Signling Technology, 95S, 1:, dilution), cleved cspse-3 (Asp175) (5A1E) rit monoclonl ntiody (Cell Signling Technology, 966S, 1: dilution), FLCN (D1G9) rit monoclonl ntiody (Cell Signling Technology, 3697S, 1:, dilution), HSP9 (C5G5) rit monoclonl ntiody no. 877(Cell Signling Technology, 877S, 1:1, dilution), GAPDH (D16H11) XP rit monoclonl ntiody (Cell Signling Technology, 517S, 1:5, dilution), p7 S6 kinse α ntiody (C-18) (Snt Cruz Biotechnology, sc-3, 1:1, dilution), SQSTM1 ntiody (H-9) (Snt Cruz Biotechnology, sc-575, 1:, dilution), LKB1 ntiody (E-9) (Snt Cruz Biotechnology, sc-3733, 1:, dilution). Sucellulr frctiontion. Cells were collected using trypsin nd wshed twice with PBS. Cell pellets were lysed in uffer I contining mm HEPES, 1 mm KCl, mm MgCl nd.5% NP. After centrifugtion, superntnts were collected s cytoplsmic lysis. Pellets were further lysed in uffer II contining.5 M NCl, mm HEPES, 1 mm KCl, mm MgCl nd.5% NP. Superntnts were collected s nucler lysis y centrifugtion. Cytoplsmic nd nucler frctions were split for RNA extrction nd rel-time PCR or protein extrction nd western lotting. HSP9 nd PARP were used s mrkers of cytoplsm nd nucleus in western lotting. GAPDH nd U1 were used s mrkers of cytoplsm nd nucleus in reltime PCR. Immunofluorescence. Cells were cultured on glss coverslips in 6-well pltes, wshed once with PBS, nd fixed in % prformldehyde for 3 min. Coverslips were mounted using Immu-mount (Thermo Shndon) nd imges were cptured using n Olympus confocl microscope. For quntifiction of utophgic cells, cells with >1 GFP LC3 punctute dots were considered positive. Positive cells ws counted nd expressed s percentge of totl utophgic cells. kinse ssy. In vitro kinse ctivity ws ssessed using the Promeg (A1/B1/G1) Kinse Enzyme System (V191) ccording to the mnufcturer s instructions. In the kinse ssy using the ACC frgment, cterilly purified GST ACC 1 13 mino cid protein ws dilysed ginst mm Tris-HCl, ph 8. nd 1% glycerol t C overnight. For kinse ssy, SFB α1, Flg β nd Flg γ1 plsmids were co-trnsfected into HEK93T cells. Cells were lysed 8 h fter trnsfection. complex ws pulled down y S protein eds nd sujected to the kinse ssy in the presence of µm cold ATP, 1 µg in vitro synthesized RNAs nd 1 µg GST fusion proteins mentioned ove. The rection mixture ws incuted t 3 C for 3 min, terminted with SDS-loding uffer nd sujected to SDS PAGE for western lot nlysis. Phosphoryltion of ACC t the Ser79 site ws determined y ACC Ser79 phospho-specific ntiody. NATURE CELL BIOLOGY 16 Mcmilln Pulishers Limited. All rights reserved

14 M E T H O D S DOI: 1.138/nc338 Xenogrft model. All niml experiments with femle thymic Nude Foxn1 nu mice (6-week-old) were performed in ccordnce with protocol pproved y the Institutionl Animl Cre nd Use Committee of MD Anderson Cncer Center, which is in full complince with policies of the Institutionl Animl Core nd Use Committee (IACUC). Animls rriving in our fcility were rndomly put into cges with five mice ech. They were implnted with respective tumour cells in the unit of cges, which were rndomly selected. MDA-MB-31 cells were counted nd suspended t ml 1 in PBS, pproximtely one million cells were injected sucutneously into the flnk of ech mouse with different genotypes, five mice per group. Tumour volume mesurement ws initited t two weeks fter injection (defined s strting time point: week ). Tumour progression ws then monitored y i-dimensionl tumour mesurements every five dys using clliper until the endpoint. Mice were euthnized t the endpoint nd the tumours were excised for further experiments. The tumour volume ws clculted ccording to the eqution v = length width 1/. The tumour volume t week n is expressed s reltive tumour volume (RTV) nd clculted ccording to the following formul: RTV = TV n /TV, where TVn is the tumour volume t week n nd TV is the tumour volume t week. The investigtors were not linded to lloction during experiments nd outcome ssessment. RNA-seq nd computtionl nlysis. RNA-seq ws performed t the Sequencing nd Non-Coding RNA Progrm t the MD Anderson Cncer Center using Applied Biosystems SOLiD Next Genertion Sequencing pltform. LifeScope v.5.1 ws used to lign the reds to the genome, generte rw counts corresponding to ech known gene (totl 3,8 genes, including,3 non-coding genes), nd clculte the RPKM (reds per kilose per million) vlues. We considered only non-coding genes tht expressed t reltively high levels (RPKM >) nd showed >- or <.5-fold chnges etween control nd tretment cells. This identified list of 17 upregulted nd 39 downregulted non-coding RNAs. Kpln Meier survivl nlysis of cncer ptients. We used dt sets of,1 rest tumours tht hd previously een profiled y Affymetrix microrry nlysis ( NBR expression (proe set ID: 7631_t) ws divided y the medin into high versus low expression. Survivl nlysis y Kpln Meier nd Cox proportionl hzrd nlysis were performed. TCGA dt nlysis. We downloded the level-3 gene expression dt for NBR from the TCGA pn-cncer project Synpse (Synpse ID: syn313) for rest (BRCA) nd kidney cncer (KIRC). We used pired Student s t-test to detect the sttisticl difference etween mtched tumour nd norml smples. We used logrnk tests to detect the overll survivl difference etween ptient groups. Accession numers. RNA-seq dt sets (786-O cells with or without glucose tretment) hve een deposited in the Gene Expression Omnius wesite with ccession code GSE7715. The dt sets used in Fig. g were generted from ref. 3. Oligonucleotide sequences, proes nd primers (forwrd nd reverse). qpcr primers for gene expression nd RIP: NBR-Forwrd: 5 -GGAGGTCTCCAGTTTCGGTA-3 NBR-Reverse: 5 -TTGATGTGTGCTTCCTGGG-3 (note tht this rel-time PCR primer set for NBR is designed to detect ll three splicing isoforms of NBR) GAPDH-Forwrd: 5 -CCATGGGGAAGGTGAAGGTC-3 GAPDH-Reverse: 5 -GAAGGGGTCATTGATGGCAAC-3 U1-Forwrd: 5 -TCCCAGGGCGAGGCTTATCCATT-3 U1-Reverse: 5 -GAACGCAGTCCCCCACTACCACAAAT-3 BRCA1-Forwrd: 5 -TGTGCTTTTCAGCTTGACACAGG-3 BRCA1-Reverse: 5 -CGTCTTTTGAGGTTGTATCCGCTG-3 Primers for RNA-pulldown ssy: NBR no. 1-Forwrd: 5 -TAATACGACTCACTATAGGG AGGGGTCCAGTT GCGGCTTAT-3 NBR no. 1-Reverse: 5 -AGTTT ACTTA CTATT GCTGA-3 NBR no. 1 Antisense-Forwrd: 5 -TAATACGACTCACTATAGGG AGTTTA CTTACTATT GCTGA-3 NBR no. 1 Antisense-Reverse: 5 -GGGTCCAGTTGCGGCTTAT-3 NBR no. -Forwrd: 5 -TAATACGACTCACTATAGGG GTTGCGGCTTAT TGCATCACA-3 NBR no. -Reverse: 5 -ACTATTGCTGATTTATTACAAAGGA-3 NBR no. Antisense-Forwrd: 5 -TAATACGACTCACTATAGGG ACTATT GCTGATTTA TTACAAAGGA-3 NBR no. Antisense-Reverse: 5 -GTTGCGGCTTATTGCATCACA-3 NBR no. 3-Forwrd: 5 -TAATACGACTCACTATAGGG AGCGGGGTTGCG GCTTATT-3 NBR no. 3-Reverse: 5 -TGGGATTGAGGAGGATCTTT-3 NBR no. 3 Antisense-Forwrd: 5 -TAATACGACTCACTATAGGG TGGGAT TGAGGAGGA TCTTT-3 NBR no. 3 Antisense-Reverse: 5 -GGGTTGCGGCTTATTGCATC-3 NBR no. T1-Forwrd: 5 -TAATACGACTCACTATAGGG GTAAAAGTTTT CATTTGATCTG AA-3 NBR no. T1-Reverse: 5 -AGTTT ACTTA CTATT GCTGA-3 NBR no. T3-Forwrd: 5 -TAATACGACTCACTATAGGG TTTGCTGAGGA TAATGGCCT-3 NBR no. T3-Reverse: 5 -AGTTT ACTTA CTATT GCTGA-3 NBR no. T-Forwrd: 5 -TAATACGACTCACTATAGGG AGGGGTCCAGT TGCGGCTTAT-3 NBR no. T-Reverse: 5 -TTCAGATCAAATGAAAACTTTTAC-3 NBR no. T5-Forwrd: 5 -TAATACGACTCACTATAGGG AGGGGTCCAGT TGCGGCTTAT-3 NBR no. T5-Reverse: 5 -CTTCCTGGGCTTCCAGCAC-3 NBR no. T6-Forwrd: 5 -TAATACGACTCACTATAGGG AGGGGTCCAGT TGCGGCTTAT-3 NBR no. T6-Reverse: 5 -AGGCCATTATCCTCAGCAAA-3 Sttistics nd reproduciility. Most experiments were repeted 3 5 times to e eligile for the indicted sttisticl nlyses, nd the dt exhiited norml distriution. There ws no estimtion of group vrition efore experiments. For gene expression nd RIP ssys, reltive quntities of gene expression level were normlized. The reltive quntities of RIP smples were normlized y individul inputs, respectively. All results re presented s men ± the stndrd devition (s.d.) of t lest three independent experiments, unless otherwise noted. Ech exct n vlue is indicted in the corresponding figure legend. Comprisons were performed using two-tiled pired Student s t-test ( P <.5, P <.1, nd P <.1), s indicted in the individul figures. For niml studies, five mice per group is the stndrd smple size for tumour xenogrft experiments, nd no sttisticl method ws used to predetermine smple size. None of the smples/nimls ws excluded from the experiment, nd the nimls were not rndomized. The investigtors were not linded to lloction during experiments nd outcome ssessment. For western lotting, representtive imges re shown. Ech of these experiments ws independently repeted 3 5 times. For survivl nlysis, the expression of NBR ws treted s inry vrint nd divided into high nd low level. Kpln Meier survivl curves were compred using the Gehn Breslow test with GrphPd Prism (GrphPd Softwre). The experiments were not rndomized. The investigtors were not linded to lloction during experiments nd outcome ssessment. 36. Lee, S. W. et l. Skp-dependent uiquitintion nd ctivtion of LKB1 is essentil for cncer cell survivl under energy stress. Mol. Cell 57, (15). 37. Lin, A. et l. The FoxO-BNIP3 xis exerts unique regultion of mtorc1 nd cell survivl under energy stress. Oncogene 33, (1). 38. Gn, B. et l. Lk1 regultes quiescence nd metolic homeostsis of hemtopoietic stem cells. Nture 68, 71 7 (1). 39. Gn, B. et l. mtorc1-dependent nd -independent regultion of stem cell renewl, differentition, nd moiliztion. Proc. Ntl Acd. Sci. USA 15, (8).. Gn, B. et l. FoxOs enforce progression checkpoint to constrin mtorc1-ctivted renl tumorigenesis. Cncer Cell 18, 7 8 (1). 1. Lin, A. et l. FoxO trnscription fctors promote AKT Ser73 phosphoryltion nd renl tumor growth in response to phrmcologicl inhiition of the PI3K-AKT pthwy. Cncer Res. 7, (1).. Gyorffy, B., Surowik, P., Budczies, J. & Lnczky, A. Online survivl nlysis softwre to ssess the prognostic vlue of iomrkers using trnscriptomic dt in non-smllcell lung cncer. PLoS ONE 8, e81 (13). 3. Gyorffy, B. et l. An online survivl nlysis tool to rpidly ssess the effect of,77 genes on rest cncer prognosis using microrry dt of 1,89 ptients. Brest Cncer Res. Tret. 13, (1). NATURE CELL BIOLOGY 16 Mcmilln Pulishers Limited. All rights reserved

15 DOI: 1.138/nc338 Supplementl Figure 1 The schemtic digrm of the genomic region of humn BRCA1 nd NBR genes with different splicing isoforms. Arrows represent the direction of trnscription Mcmilln Pulishers Limited. All rights reserved

16 Compound C (µm) Glucose (mm) si Glucose (mm) p- p- Supplementl Figure inctivtion y compound C or α sirna tretment. () MDA-MB-31 cells were treted with μm Compound C in or mm glucose-contining medium for hours, nd then sujected to Western lotting nlysis to mesure ctivtion. () MDA-MB-31 cells trnsfected with α or control (Ctrl) sirna were cultured in or mm glucose-contining medium for hours, nd then sujected to Western lotting nlysis to mesure ctivtion. Unprocessed originl scns of lots re shown in Supplementl Fig Mcmilln Pulishers Limited. All rights reserved

17 Ctrl sh (Glucose mm) GFP-LC3 DAPI Merge Ctrl sh Glucose (mm) p-ulk1 (Ser757) NBR sh1 NBR sh 1 Ctrl sh (Glucose mm) p-ulk1 (Ser555) 1 ULK1 1 NBR sh1 (Glucose mm) p6 NBR sh1 (Glucose mm) c 786-O d MDA-MB-31 Reltive cell numers Control shrna NBR shrna1 NBR shrna p=1.6e- Reltive cell numers Control shrna NBR shrna1 NBR shrna p=.e Dys. 1mM Glucose Medium 1 3 Dys Supplementl Figure 3 NBR knockdown ffects utophgy nd cell prolifertion in response to energy stress. () The effect of NBR deficiency on GFP-LC3 punct formtion. 786-O cells infected with either control shrna or NBR shrna were trnsfected with GFP-LC3 plsmid, nd then cultured in or mm glucose-contining medium for 18 hours. GFP-LC3 punctte foci were then detected using fluorescence microscopy. (Scle rs, µm) () The effect of NBR deficiency on ULK1 phosphoryltion nd p6 degrdtion in response to glucose strvtion. MDA-MB-31 cells infected with either control shrna or NBR shrna were cultured in or mm glucose-contining medium for 1h. Cell lystes were then nlyzed y Western lotting. (c, d) Cells infected with either control shrna or NBR shrna were cultured in 1mM glucose-contining medium for different dys s indicted, nd then sujected to cell prolifertion nlysis (Men ± s.d., n=3 iologiclly independent extrcts, two-tiled pired Student s t-test). Source dt for c, d cn e found in Supplementry Tle 1. Unprocessed originl scns of lots re shown in Supplementl Fig Mcmilln Pulishers Limited. All rights reserved

18 NBR #1 p-acc EV T T5 FL Input EV #1 lgg IP LKB1 IP Folliculin IP EV #1 EV #1 EV #1 ACC p- LKB1 p-s6k Folliculin S6K 75 HEK93T c lgg IP LKB1 IP WCL Ctrl sh NBR sh1 NBR sh Ctrl sh NBR sh1 NBR sh Ctrl sh NBR sh1 G: NBR sh LKB1 d Hel EV Lk1 EV #1 EV #1 p- LKB1 Supplementl Figure Mechnistic studies of NBR regultion of. () Protein lystes were prepred from HEK93T cells trnsfected with empty vector (EV), NBR full length (FL), T, or T5 frgment expression vectors, nd nlyzed y Western lotting s indicted. () Protein lystes prepred from UMRC cells stly expressing EV or NBR #1 expression vectors were immunoprecipitted y IgG, LKB1 or Folliculin ntiodies, nd then were nlyzed y Western lotting s indicted. Aliquots of the protein lystes (input) were lso nlyzed directly. (c) 786-O cells infected with either control shrna or NBR shrna were cultured in medium contining or mm glucose for 1 hours. Protein lystes were prepred nd immunoprecipitted y IgG or LKB1 ntiodies, nd then were nlyzed y Western lotting s indicted. Aliquots of the protein lystes (input) were lso nlyzed directly. (d) Empty vector (EV) or LKB1-infected Hel cells were trnsfected with EV or NBR #1 expression vectors. Protein lystes were prepred nd nlyzed y Western lotting s indicted. Unprocessed originl scns of lots re shown in Supplementl Fig Mcmilln Pulishers Limited. All rights reserved

19 Supplementl Figure 5 The working model of the reciprocl regultion etween NBR nd under energy stress, nd its relevnce to cncer development. See discussion for detiled description Mcmilln Pulishers Limited. All rights reserved

20 Glucose Ctrl sh NBR sh1 Strvtion (Hr): p- p-acc ACC Reltive NBR levels Glucose Strvtion (Hr): p=3.6e-3 p=1.1e- 8 1 Supplementl Figure 6 NBR deficiency ffects ctivtion under long periods of energy stress. () MDA-MB-31 cells infected with either control shrna or NBR shrna were cultured in mm glucose-contining medium for different hours, nd protein lystes were prepred nd nlyzed y Western lotting. () MDA-MB-31 cells were cultured in mm glucosecontining medium for different hours, nd then sujected to rel-time PCR nlysis to mesure NBR expression (Men ± s.d., n=3 iologiclly independent extrcts, two-tiled pired Student s t-test). Source dt for cn e found in Supplementry Tle 1. Unprocessed originl scns of lots re shown in Supplementl Fig Mcmilln Pulishers Limited. All rights reserved

21 Reltive BRCA1 levels mm Glucose mm Glucose Glucose (mm) BRCA1 Vinculin Ctrl sh NBR sh1 NBR sh 1. Ctrl sh NBR sh1 NBR sh Supplementl Figure 7 NBR deficiency does not ffect BRCA1 expression. () MDA-MB-31 cells infected with either control shrna or NBR shrna were cultured in or mm glucose-contining medium for hours, nd then sujected to rel-time PCR nlysis to mesure BRCA1 expression (Men ± s.d., n=3 iologiclly independent extrcts, two-tiled pired Student s t-test). () Cell lystes were lso nlyzed y Western lotting s indicted. Source dt for cn e found in Supplementry Tle 1. Unprocessed originl scns of lots re shown in Supplementl Fig Mcmilln Pulishers Limited. All rights reserved

22 Fig. 1d Supplementl Figure 8 Unprocessed scns of full lots Mcmilln Pulishers Limited. All rights reserved

23 Fig. Fig. c Supplementl Figure 8 continued Mcmilln Pulishers Limited. All rights reserved

24 Fig. d Fig. e Supplementl Figure 8 continued Mcmilln Pulishers Limited. All rights reserved

25 Fig. 3f Fig. d Supplementl Figure 8 continued Mcmilln Pulishers Limited. All rights reserved

26 Fig. 5 d c e H f Supplementl Figure 8 continued Mcmilln Pulishers Limited. All rights reserved

27 Fig. 6 d e g Supplementl Figure 8 continued Mcmilln Pulishers Limited. All rights reserved

28 Fig. 7 c f Supplementl Figure 8 continued Mcmilln Pulishers Limited. All rights reserved

29 Fig. S Fig. S3 Supplementl Figure 8 continued Mcmilln Pulishers Limited. All rights reserved

30 Fig. S c d Supplementl Figure 8 continued Mcmilln Pulishers Limited. All rights reserved

31 Fig. S6 Fig. S7 Supplementl Figure 8 continued Mcmilln Pulishers Limited. All rights reserved