#METABOLOMICS WORKBENCH RCulpHill_20210317_102457 DATATRACK_ID:2534 STUDY_ID:ST001730 ANALYSIS_ID:AN002815 PROJECT_ID:PR001108
VERSION             	1
CREATED_ON             	March 24, 2021, 2:54 pm
#PROJECT
PR:PROJECT_TITLE                 	Mitochondrial ATP fuels ABC transporter-mediated drug efflux in cancer
PR:PROJECT_TITLE                 	chemoresistance
PR:PROJECT_SUMMARY               	Chemotherapy remains the standard of care for most cancers worldwide, however
PR:PROJECT_SUMMARY               	development of chemoresistance due to the presence of the drug-effluxing ABC
PR:PROJECT_SUMMARY               	transporters remains a significant problem. The development of safe and
PR:PROJECT_SUMMARY               	effective means to overcome chemoresistance is critical for achieving durable
PR:PROJECT_SUMMARY               	remissions in many cancer patients. We have investigated the energetic demands
PR:PROJECT_SUMMARY               	of ABC transporters in the context of the metabolic adaptations of
PR:PROJECT_SUMMARY               	chemoresistant cancer cells. Here we show that ABC transporters use
PR:PROJECT_SUMMARY               	mitochondrial-derived ATP as a source of energy to efflux drugs out of cancer
PR:PROJECT_SUMMARY               	cells. We further demonstrate that the loss of MCJ (DnaJC15), an endogenous
PR:PROJECT_SUMMARY               	negative regulator of mitochondrial respiration, in chemoresistant cancer cells
PR:PROJECT_SUMMARY               	boosts their ability to produce ATP from mitochondria and fuel ABC transporters.
PR:PROJECT_SUMMARY               	We have developed novel MCJ mimetics that can attenuate mitochondrial
PR:PROJECT_SUMMARY               	respiration and safely overcome chemoresistance in vitro and in vivo.
PR:PROJECT_SUMMARY               	Administration of MCJ mimetics in combination with standard chemotherapeutic
PR:PROJECT_SUMMARY               	drugs could therefore become an new strategy for treatment of multiple cancers.
PR:INSTITUTE                     	University of Colorado Denver
PR:DEPARTMENT                    	Biochemistry and Molecular Genetics
PR:LABORATORY                    	Angelo D'Alessandro
PR:LAST_NAME                     	Culp-Hill
PR:FIRST_NAME                    	Rachel
PR:ADDRESS                       	12801 E 17th Ave L18-9403D, Aurora, Colorado, 80045, USA
PR:EMAIL                         	rachel.hill@cuanschutz.edu
PR:PHONE                         	303-724-5798
#STUDY
ST:STUDY_TITLE                   	Mitochondrial ATP fuels ABC transporter-mediated drug efflux in cancer
ST:STUDY_TITLE                   	chemoresistance
ST:STUDY_SUMMARY                 	Chemotherapy remains the standard of care for most cancers worldwide, however
ST:STUDY_SUMMARY                 	development of chemoresistance due to the presence of the drug-effluxing ABC
ST:STUDY_SUMMARY                 	transporters remains a significant problem. The development of safe and
ST:STUDY_SUMMARY                 	effective means to overcome chemoresistance is critical for achieving durable
ST:STUDY_SUMMARY                 	remissions in many cancer patients. We have investigated the energetic demands
ST:STUDY_SUMMARY                 	of ABC transporters in the context of the metabolic adaptations of
ST:STUDY_SUMMARY                 	chemoresistant cancer cells. Here we show that ABC transporters use
ST:STUDY_SUMMARY                 	mitochondrial-derived ATP as a source of energy to efflux drugs out of cancer
ST:STUDY_SUMMARY                 	cells. We further demonstrate that the loss of MCJ (DnaJC15), an endogenous
ST:STUDY_SUMMARY                 	negative regulator of mitochondrial respiration, in chemoresistant cancer cells
ST:STUDY_SUMMARY                 	boosts their ability to produce ATP from mitochondria and fuel ABC transporters.
ST:STUDY_SUMMARY                 	We have developed novel MCJ mimetics that can attenuate mitochondrial
ST:STUDY_SUMMARY                 	respiration and safely overcome chemoresistance in vitro and in vivo.
ST:STUDY_SUMMARY                 	Administration of MCJ mimetics in combination with standard chemotherapeutic
ST:STUDY_SUMMARY                 	drugs could therefore become an new strategy for treatment of multiple cancers.
ST:INSTITUTE                     	University of Colorado Denver
ST:DEPARTMENT                    	Biochemistry and Molecular Genetics
ST:LABORATORY                    	Angelo D'Alessandro
ST:LAST_NAME                     	Culp-Hill
ST:FIRST_NAME                    	Rachel
ST:ADDRESS                       	12801 E 17th Ave L18-9403D, Aurora, Colorado, 80045, USA
ST:EMAIL                         	rachel.hill@cuanschutz.edu
ST:PHONE                         	303-724-5798
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	-	nci-adr-res-1	line:NCI-ADR-RES	RAW_FILE_NAME=nci-adr-res-1
SUBJECT_SAMPLE_FACTORS           	-	nci-adr-res-2	line:NCI-ADR-RES	RAW_FILE_NAME=nci-adr-res-2
SUBJECT_SAMPLE_FACTORS           	-	nci-adr-res-3	line:NCI-ADR-RES	RAW_FILE_NAME=nci-adr-res-3
SUBJECT_SAMPLE_FACTORS           	-	nci-adr-res-4	line:NCI-ADR-RES	RAW_FILE_NAME=nci-adr-res-4
SUBJECT_SAMPLE_FACTORS           	-	ovcar8-1	line:OVCAR8	RAW_FILE_NAME=ovcar8-1
SUBJECT_SAMPLE_FACTORS           	-	ovcar8-2	line:OVCAR8	RAW_FILE_NAME=ovcar8-2
SUBJECT_SAMPLE_FACTORS           	-	ovcar8-3	line:OVCAR8	RAW_FILE_NAME=ovcar8-3
SUBJECT_SAMPLE_FACTORS           	-	ovcar8-4	line:OVCAR8	RAW_FILE_NAME=ovcar8-4
#COLLECTION
CO:COLLECTION_SUMMARY            	NCI/ADR-RES (formerly MCF7/Adr) were previously described. NCI/ADR-RES cells
CO:COLLECTION_SUMMARY            	were verified to be of ovarian origin by genotyping. OVCAR-8 cells provided by
CO:COLLECTION_SUMMARY            	Dr. Ernst Lengyel at the University of Chicago. All cancer cell lines were
CO:COLLECTION_SUMMARY            	maintained in RPMI 1640 (Sigma R8758) containing glucose (2 mg/ml) and glutamine
CO:COLLECTION_SUMMARY            	(0.6 mg/ml) but no pyruvate and was supplemented with 5% Fetal calf serum.
CO:SAMPLE_TYPE                   	Cultured cells
#TREATMENT
TR:TREATMENT_SUMMARY             	N/A - compared cell lines only
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	All cancer cell lines were maintained in RPMI 1640 (Sigma R8758) containing
SP:SAMPLEPREP_SUMMARY            	glucose (2 mg/ml) and glutamine (0.6 mg/ml) but no pyruvate and was supplemented
SP:SAMPLEPREP_SUMMARY            	with 5% Fetal calf serum. Metabolic profile comparisons of OVCAR-8 and
SP:SAMPLEPREP_SUMMARY            	NCI/ADR-RES cells were performed on equal cell numbers. Cells were cultured
SP:SAMPLEPREP_SUMMARY            	under normal conditions, detached using trypsin-EDTA (0.05 %), counted,
SP:SAMPLEPREP_SUMMARY            	normalized to 0.5 x 10e6 in each sample, and cell pellets were snap frozen in
SP:SAMPLEPREP_SUMMARY            	liquid nitrogen prior to analysis. Briefly, 2 x 10e6 cells and 20μL cell media
SP:SAMPLEPREP_SUMMARY            	were extracted in 1mL and 980μL cold lysis and extraction buffer (methanol :
SP:SAMPLEPREP_SUMMARY            	acetonitrile : water, 5:3:2), respectively, and pellets were discarded.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Positive Mode 10 μL of water and methanol soluble fractions were run through a
CH:CHROMATOGRAPHY_SUMMARY        	Kinetex C18 1.7 μm, 100 x 2.1 mm (Phenomenex) reversed phase column (Positive
CH:CHROMATOGRAPHY_SUMMARY        	ion mode - phase A: water, 0.1 % formic acid; B: acetonitrile, 0.1 % formic
CH:CHROMATOGRAPHY_SUMMARY        	acid) via an ultra-high performance chromatographic system (UHPLC - Vanquish,
CH:CHROMATOGRAPHY_SUMMARY        	Thermo Fisher). UHPLC was coupled in line with a high-resolution quadrupole
CH:CHROMATOGRAPHY_SUMMARY        	Orbitrap instrument run in positive polarity mode (QExactive, Thermo Fisher) at
CH:CHROMATOGRAPHY_SUMMARY        	70,000 resolution (at 200 m/z). Gradients and other technical parameters and the
CH:CHROMATOGRAPHY_SUMMARY        	variants employed herein have been extensively described.
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Vanquish
CH:COLUMN_NAME                   	Phenomenex Kinetex C18 (150 x 2.1mm, 2.6 um)
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	UHPLC was coupled in line with a high-resolution quadrupole Orbitrap instrument
MS:MS_COMMENTS                   	run in positive polarity mode (QExactive, Thermo Fisher) at 70,000 resolution
MS:MS_COMMENTS                   	(at 200 m/z). Gradients and other technical parameters and the variants employed
MS:MS_COMMENTS                   	herein have been extensively described.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	Relative Abundance
MS_METABOLITE_DATA_START
Samples	nci-adr-res-1	nci-adr-res-2	nci-adr-res-3	nci-adr-res-4	ovcar8-1	ovcar8-2	ovcar8-3	ovcar8-4
Factors	line:NCI-ADR-RES	line:NCI-ADR-RES	line:NCI-ADR-RES	line:NCI-ADR-RES	line:OVCAR8	line:OVCAR8	line:OVCAR8	line:OVCAR8
L-alanine	24132900	24873720	25174420	23038700	21141530	21547460	22410700	25334360
L-arginine	26149860	24809140	24032850	24282840	21107350	18888230	21851600	19391900
L-asparagine	5376476	5445190	5883608	5443122	3440691	4321256	4403402	5070458
L-aspartate	35179980	35806660	40819640	38864620	26279810	31485470	30998100	34204950
L-cysteine	4718016	5363604	5676248	5379934	4783919	5317896	5112026	7107130
L-glutamate	267024100	292997000	287993100	287255300	272885900	310640000	304423700	370463000
L-glutamine	52580140	53974240	49805170	49857340	49221210	47953230	57661300	60530610
glycine	3541181	3333057	3321975	3718810	3051076	3519917	3764035	4425668
L-histidine	4421904	4477788	4884954	4610672	2680365	2617757	3308872	3275711
L-leucine/isoleucine	113255000	118609000	124899700	127929700	74242110	96083810	89397830	101395800
L-lysine	8384514	8156290	8980707	9842918	3878874	4772962	5301782	5412290
L-methionine	5549216	6063790	5646764	7028622	3650989	3707641	5035878	5368840
L-phenylalanine	5166852	5170816	5847400	6412360	2960734	3473377	4284468	4177542
L-proline	150354000	130625300	151071800	150626500	152427400	149005300	158140800	171619900
L-serine	2022088	2253140	2246038	2767568	1284620	1389257	1716988	1721470
L-threonine	7492732	7684656	8142142	7698170	4824012	5310648	6411840	7024102
L-tryptophan	11016400	9937601	11008610	13357970	7955984	7393658	10201120	9179329
L-tyrosine	5987270	6410512	6459076	7294688	3006245	3898670	4757924	5057406
L-valine	52200230	52468340	56092430	52309200	40083990	37990830	43466040	41210150
ATP	3185185	2550247	2447309	2065439	387825	585775	712324	503001
AMP	21303820	19382830	17768540	14960530	740199	837652	1008284	812021
dAMP	247010	363603	311954	314133	257605	305570	338418	332865
GDP	1396264	1410311	1303427	1315924	358284	397962	432944	292844
GMP	2400772	2390090	2258350	1905687	95866	125174	120159	89576
Guanine	403299	364632	427807	548226	1743644	1936989	2431683	2293858
Uracil	5121742	6437090	7435372	7724004	9321921	9244679	11099060	9295071
IMP	496053	504205	499743	401931	23723	22957	25811	26859
Xanthine	6570614	6918538	8159514	9523261	12584400	12539520	14020030	16673250
(S)(+)-Allantoin	149478	143268	132423	154686	121901	134260	142152	121919
Urate	31543	34853	34968	38424	16682	23249	24859	28027
5'-Phosphoribosyl-N-formylglycinamide	393282	349770	421461	382274	209665	208253	304488	253735
Nicotinamide	28843990	32332140	35287610	33250980	14017730	16460550	15604500	18348710
Adenylosuccinic acid	592185	432131	468561	409512	17051	22110	17653	19799
NADPH	56418	36413	44921	47213	6419	27721	30934	38429
NAD+	3711520	4566386	4461792	3716853	1893804	2314302	2526449	2518169
NADH	220557	177386	158473	163799	214669	219590	181276	212269
Glutathione	334475500	312559800	399524000	311638700	270485600	277005400	335149100	377691900
Glutathione disulfide	562418	670356	651173	591421	400096	500272	571475	470018
Cys-Gly	4778120	5860486	6080250	5368502	3963465	4502258	4801410	5232628
(5-L-Glutamyl)-L-glutamine	298910	339812	401673	419419	1203147	1277576	1472158	1753495
L-Homocysteine	56438	68034	65424	59196	16893	27312	30072	25626
Cystathionine	3064252	3084263	3290207	3202434	939436	1103815	1054236	1513800
Dimethylglycine	1006958	970756	949508	1005867	1064278	1100636	1159471	1134494
Phosphoserine	1322338	1487435	1617949	1487417	3348231	3353152	2480110	2747266
S-Adenosyl-L-methionine	857157	900380	686430	753075	361025	493031	666497	612978
Ornithine	662982	553467	816176	1204150	27990	32906	89827	45578
L-Citrulline	3890313	4907072	4826986	5106004	1511116	1599510	2043886	2401423
Putrescine	2599988	2593844	2425202	2188696	2173933	2563768	2555965	2347354
Spermidine	2828642	2803390	2409113	2258311	3533436	4818542	4658502	4082872
Spermine	533013	639647	607829	603933	624735	1409646	1780039	924925
Creatine	1402299000	1353976000	1437415000	1457863000	1142794000	1183287000	1175427000	1219381000
Creatinine	9426684	9226068	9902754	9629811	6546842	7579214	7576200	6862650
5-6-Dihydrouracil	763100	783762	799960	751920	602312	632448	668818	655418
D-4--Phosphopantothenate	287645	262349	321434	312533	238777	211419	177671	207363
Taurine	15150680	17783000	17762800	17329630	33390170	37861410	39300040	39711070
Hypotaurine	200338	215927	213905	186905	338007	387432	335134	337774
5-Hydroxyindoleacetate	1631244	1717060	1833503	1828822	744468	736437	941529	923388
Indole	20229	16059	17236	20156	13080	12980	18225	18300
Indole-3-acetate	145789	128202	131939	138109	102711	89752	108079	109222
quinolinic acid	49803	35243	41481	58257	95787	101460	100626	78736
Choline	374082000	363116700	396306000	359328400	355437100	367307100	379956100	411267000
L-Carnitine	10135310	11225300	10895600	10845910	10978970	11315690	10960480	13822620
acetyl-carnitine (acyl-C2)	2721396	2456492	2619687	1919489	4820470	4669122	4351248	3549925
propionyl-carnitine (acyl-C3)	538223	468731	550130	451523	1730252	1894852	1917165	1242270
butanoyl-l-carnitine (acyl-C4)	608231	426179	542131	448173	2085816	1921537	2229998	1764324
hexanoyl-L-carnitine (acyl-C6)	187820	184197	157234	141867	206256	175590	168260	161773
O-tetradecanoyl-L-carnitine (acyl-C14)	709504	601820	360718	413932	76262	1033240	1065120	1108112
Hexadecenoyl-carnitine (acyl-C16:1)	236809	106778	80842	90218	37053	301541	356367	364068
O-octadecenoyl-L-carnitine (acyl-C18:1)	484723	208805	180566	126465	61318	692775	553724	713094
acyl-C18:2 (Linoleoyl-CoA)	541926	158983	120888	98731	50952	939122	1230528	1510386
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	kegg	mass	rt
L-alanine	C00041	90.05479	0.6487375
L-arginine	C00062	175.1187	0.6238049
L-asparagine	C00152	133.0605	0.6569
L-aspartate	C00049	134.0446	0.6527199
L-cysteine	C00097	122.0269	0.6616744
L-glutamate	C00025	148.0603	0.6512074
L-glutamine	C00064	147.0762	0.6524274
glycine	C00037	76.03923	0.644645
L-histidine	C00135	156.0767	0.595175
L-leucine/isoleucine	C00123	132.1018	0.6804075
L-lysine	C00047	147.1126	0.59813
L-methionine	C00073	150.0582	0.6771668
L-phenylalanine	C00079	166.086	0.6771901
L-proline	C00148	116.0704	0.66444
L-serine	C00065	106.0498	0.6435924
L-threonine	C00188	120.0654	0.6580949
L-tryptophan	C00078	205.0968	1.721723
L-tyrosine	C00082	182.081	0.67885
L-valine	C00183	118.086	0.6588225
ATP	C00002	505.9882	0.7575678
AMP	C00020	346.0556	0.6726601
dAMP	C00360	332.0773	0.6958891
GDP	C00035	442.0168	0.6207583
GMP	C00144	362.0507	0.6581243
Guanine	C00242	152.0542	1.214629
Uracil	C00106	113.0344	0.680365
IMP	C00130	347.0402	0.6598513
Xanthine	C00385	153.0406	0.6771919
(S)(+)-Allantoin	C02350	157.0355	0.6661596
Urate	C00366	167.0201	0.6772898
5'-Phosphoribosyl-N-formylglycinamide	C04376	315.0577	0.691133
Nicotinamide	C00153	123.0551	0.6759975
Adenylosuccinic acid	C03794	464.0803	0.6821898
NADPH	C00005	745.9999	1.165878
NAD+	C00003	664.1149	0.6627129
NADH	C00004	666.1302	0.7354685
Glutathione	C00051	308.0901	0.67784
Glutathione disulfide	C00127	613.158	0.6781725
Cys-Gly	C01419	179.0482	0.6782131
(5-L-Glutamyl)-L-glutamine	C05283	276.1183	0.6559027
L-Homocysteine	C00155	136.0428	0.6758068
Cystathionine	C00542	223.0741	0.641854
Dimethylglycine	C01026	104.0705	0.6613175
Phosphoserine	C02532	186.0162	0.6469325
S-Adenosyl-L-methionine	C00019	399.1436	0.600277
Ornithine	C01602	133.0971	0.6010924
L-Citrulline	C00327	176.1028	0.6514632
Putrescine	C00134	89.10718	0.5888
Spermidine	C00315	146.165	0.6001925
Spermine	C00750	203.2227	0.6069204
Creatine	C00300	132.0766	0.65541
Creatinine	C00791	114.066	0.6490426
5-6-Dihydrouracil	C00429	115.0501	0.6762049
D-4--Phosphopantothenate	C03492	298.0692	0.6651585
Taurine	C00245	126.0218	0.6552325
Hypotaurine	C00519	110.0268	0.6532136
5-Hydroxyindoleacetate	C05635	190.0533	0.7137606
Indole	C00463	118.065	1.72847
Indole-3-acetate	C00954	174.0582	1.726332
quinolinic acid	C03722	166.0166	0.6680105
Choline	C00114	104.1075	0.6660002
L-Carnitine	C00318	162.1123	0.652765
acetyl-carnitine (acyl-C2)	C02571	204.1227	0.6777237
propionyl-carnitine (acyl-C3)	C03017	218.1383	0.6789312
butanoyl-l-carnitine (acyl-C4)	C02862	232.1539	0.6786808
hexanoyl-L-carnitine (acyl-C6)	HMDB00756	260.1851	1.770188
O-tetradecanoyl-L-carnitine (acyl-C14)	HMDB05066	372.3101	1.999754
Hexadecenoyl-carnitine (acyl-C16:1)	HMDB06317	398.3261	2.010351
O-octadecenoyl-L-carnitine (acyl-C18:1)	HMDB06351	426.3569	2.088172
acyl-C18:2 (Linoleoyl-CoA)	HMDB01064	424.3411	2.029301
METABOLITES_END
#END