#METABOLOMICS WORKBENCH RCulpHill_20210318_135702_mwtab.txt DATATRACK_ID:2535 STUDY_ID:ST001731 ANALYSIS_ID:AN002818 PROJECT_ID:000000
VERSION             	1
CREATED_ON             	March 24, 2021, 2:58 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 (part-II)
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           	-	control1	Factor:Control	RAW_FILE_NAME=control1
SUBJECT_SAMPLE_FACTORS           	-	control2	Factor:Control	RAW_FILE_NAME=control2
SUBJECT_SAMPLE_FACTORS           	-	control3	Factor:Control	RAW_FILE_NAME=control3
SUBJECT_SAMPLE_FACTORS           	-	control4	Factor:Control	RAW_FILE_NAME=control4
SUBJECT_SAMPLE_FACTORS           	-	siMCJ1	Factor:siMCJkd	RAW_FILE_NAME=siMCJ1
SUBJECT_SAMPLE_FACTORS           	-	siMCJ2	Factor:siMCJkd	RAW_FILE_NAME=siMCJ2
SUBJECT_SAMPLE_FACTORS           	-	siMCJ3	Factor:siMCJkd	RAW_FILE_NAME=siMCJ3
SUBJECT_SAMPLE_FACTORS           	-	siMCJ4	Factor:siMCJkd	RAW_FILE_NAME=siMCJ4
#COLLECTION
CO:COLLECTION_SUMMARY            	MCF7 cells were purchased from American Type Culture Collection (ATCC). All
CO:COLLECTION_SUMMARY            	cancer cell lines were maintained in RPMI 1640 (Sigma R8758) containing glucose
CO:COLLECTION_SUMMARY            	(2 mg/ml) and glutamine (0.6 mg/ml) but no pyruvate and was supplemented with 5%
CO:COLLECTION_SUMMARY            	Fetal calf serum.
CO:SAMPLE_TYPE                   	Cultured cells
#TREATMENT
TR:TREATMENT_SUMMARY             	MCF7 cell transfections were performed using siPORT NeoFX Transfection Agent
TR:TREATMENT_SUMMARY             	(ThermoFisher Scientific) following the recommended protocol for an
TR:TREATMENT_SUMMARY             	oligonucleotide siRNA based on the previously described MCJ targeting sequence.
#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 cells were performed
SP:SAMPLEPREP_SUMMARY            	on equal cell numbers. Cells were cultured under normal conditions, detached
SP:SAMPLEPREP_SUMMARY            	using trypsin-EDTA (0.05 %), counted, normalized to 0.5 x 10e6 in each sample,
SP:SAMPLEPREP_SUMMARY            	and cell pellets were snap frozen in liquid nitrogen prior to analysis. Briefly,
SP:SAMPLEPREP_SUMMARY            	2 x 10e6 cells and 20μL cell media were extracted in 1mL and 980μL cold lysis
SP:SAMPLEPREP_SUMMARY            	and extraction buffer (methanol : acetonitrile : water, 5:3:2), respectively,
SP:SAMPLEPREP_SUMMARY            	and pellets were discarded.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Negative 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 (Negative
CH:CHROMATOGRAPHY_SUMMARY        	ion mode – phase A: 1 mM NH4Ac 95:5 water : acetonitrile; phase B: 1 mM NH4Ac
CH:CHROMATOGRAPHY_SUMMARY        	95:5 acetonitrile : water) via an ultra-high performance chromatographic system
CH:CHROMATOGRAPHY_SUMMARY        	(UHPLC - Vanquish, Thermo Fisher). UHPLC was coupled in line with a
CH:CHROMATOGRAPHY_SUMMARY        	high-resolution quadrupole Orbitrap instrument run in negative polarity mode
CH:CHROMATOGRAPHY_SUMMARY        	(QExactive, Thermo Fisher) at 70,000 resolution (at 200 m/z). Gradients and
CH:CHROMATOGRAPHY_SUMMARY        	other technical parameters and the variants employed herein have been
CH:CHROMATOGRAPHY_SUMMARY        	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                      	NEGATIVE
MS:MS_COMMENTS                   	UHPLC was coupled in line with a high-resolution quadrupole Orbitrap instrument
MS:MS_COMMENTS                   	run in negative 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	control1	control2	control3	control4	siMCJ1	siMCJ2	siMCJ3	siMCJ4
Factors	Factor:Control	Factor:Control	Factor:Control	Factor:Control	Factor:siMCJkd	Factor:siMCJkd	Factor:siMCJkd	Factor:siMCJkd
ATP	1254300	1530584	995836	958442	1172597	860470	890423	779232
ADP	6062622	7955310	5602974	5072800	7228896	6342440	6842992	6700468
AMP	32556540	31746980	29555320	22912970	32130200	27251380	24645710	28246100
GTP	124908	136036	71206	57904	71946	55728	45124	44497
GDP	566391	731503	441869	417703	605072	518753	584348	565662
GMP	5863058	5720972	5323990	4369844	5765728	4915186	5296294	5309808
Guanosine	184637	200140	224381	230934	198290	202758	210254	234991
Cytidine	4258840	4081814	3979304	3723048	3679043	3794888	4158999	4078820
dTMP	411511	429250	485030	352213	483114	463847	380226	458784
Thymine	372827	355949	339870	304087	408072	362376	392651	421047
UDP	2405901	2709062	2410566	2182940	3024296	2483881	3412353	3136296
UMP	18035190	18319170	15641880	13280450	19345150	14814690	16090320	17774450
Inosine	2169625	1967127	2384894	2400882	2238472	2021310	2558873	2838437
Hypoxanthine	6860700	7321158	6882526	6655866	7745394	6562548	8191108	8814087
Xanthine	117782	113028	117431	98068	123021	89240	131549	136834
(S)(+)-Allantoin	98624	155866	111863	117258	146701	101435	159993	145454
3',5'-Cyclic AMP	380352	330791	273790	330435	216173	228182	241637	326333
5'-Phosphoribosyl-N-formylglycinamide	1006487	1064766	813768	799089	859778	661648	863961	884149
5-6-Dihydrothymine	5289740	5171485	5010136	4178330	5552880	4710194	5459394	5261210
Pyridoxal	77857	95526	91445	95018	177061	131465	167540	139187
UDP-glucose	3653380	3493188	2978648	2779505	3249187	2494146	2863122	2477913
Diphosphate	11289200	11573440	11066300	10486700	10877330	9102285	10798810	11402520
D-Glucose	313115	361059	362209	405710	634458	595383	636154	663155
D-Fructose 1-6-bisphosphate	804793	840657	737929	678194	770357	736081	844272	723142
2/3-Phospho-D-glycerate	371953	437780	577738	675923	502133	485860	565517	445766
Pyruvate	857692	586692	712240	597797	705659	511209	678480	707869
Lactate	99169860	103555700	100624600	86221250	107050900	85463360	106173000	109132600
Mannitol	358611	356649	1694934	1320946	481049	362446	814153	572011
D-Arabitol	84435	88475	82179	74284	111884	93793	123191	141977
Citrate	16947670	15848090	14117900	13776620	13266000	11757570	13457180	13584280
2-Oxoglutarate	75375	77974	93350	89229	78770	64490	75362	81925
Succinate	13116930	15193850	11575270	11157310	13738470	11741650	14665800	15966020
Fumarate	2576042	2734026	2415042	2376355	2985058	2251292	3186203	3329032
Malate	20146110	19592960	17385440	16860250	22124660	17198740	24288390	26901990
2-Hydroxyglutarate/Citramalate	210640	197732	168370	191753	187260	163526	206275	230365
6-Phospho-D-gluconate	295199	191750	246602	235570	306045	229841	272630	314959
alpha-D-Ribose 1-phosphate	1277626	1163309	1256278	1355930	1008270	1137660	1211630	1351017
5-Oxoproline	25693300	24988140	22985370	22522250	27009610	21710950	25953930	25440620
S-Glutathionyl-L-cysteine	54479	129703	117643	92305	205193	199424	296115	262389
Ascorbate	497502	594698	502170	502201	735509	556435	756336	732785
Dehydroascorbate	256522	281027	230112	252942	251598	210344	223946	222209
gamma-L-Glutamyl-D-alanine	960131	778232	868993	878824	875188	783620	1003342	878359
Dimethylglycine	1154989	1137117	982330	1015306	1187938	1042712	1212398	1362486
Argininosuccinate	982536	1165253	915336	873787	1128066	941351	1141313	1113643
UDP-N-acetyl-D-glucosamine	24491450	23210390	21126130	18737620	24766050	18033610	22331370	18059450
CMP-N-acetylneuraminate	86112	90328	77382	71631	112023	66512	99514	79291
N-Acetylornithine	125452	133242	100055	111005	98479	115803	122869	133571
trans-4-Hydroxy-L-proline	4872758	4482142	4430822	4105438	5598324	4854798	5688126	5104546
Pantothenol	1362851	1488301	996820	875921	1211968	982701	1397303	1160601
5-6-Dihydrouracil	1110577	1039200	976594	763332	1046147	1062221	1052752	1023356
L-Methionine S-oxide	55844	41614	30716	42270	66012	44709	51428	26496
5-Hydroxyindoleacetate	253555	230227	198427	203415	257974	200634	269094	273664
Indole	29775	27821	28361	26927	36856	30704	35785	34315
quinolinic acid	397788	459096	374195	329539	454323	354074	458894	485588
g-Oxalo-crotonate	65875	71575	90277	82450	99906	84382	113278	109916
Glycerol 3-phosphate	613026	867648	540304	574563	829118	549075	783487	815413
Ethanolamine phosphate	4859090	4268548	4652242	4026841	6680066	5494050	6257162	6691882
N-Methylethanolamine phosphate	598252	510990	614603	506422	700694	496993	647677	667469
sn-glycero-3-Phosphoethanolamine	16993300	16567120	14804800	13322880	17145990	15428490	12634840	15733880
CDP-choline	82260	107749	97313	121942	159146	106890	180757	203211
Pentanoate (valerate)	35819	39556	40807	47239	68585	65924	80118	86424
Hexanoic acid (caproate)	101152	128391	164320	166387	275949	287758	308075	325484
Cholesterol sulfate	2946209	1880575	2905510	4511776	6379068	3975874	5950348	7280414
Glycochenodeoxycholic acid	8750	13280	12094	8935	9303	12091	9220	11288
Tauroursodeoxycholic/taurochenodeoxycholic/taurodeoxycholic acid	21766	37584	26999	25254	22329	29121	22541	19325
S-Acylglutathione	207225	264194	258222	191920	205525	204185	275029	269634
Oxalosuccinate	8345	12707	17996	22311	38096	31260	49171	53897
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	CmpdID	parent	medRt
ATP	C00002	505.9879	0.62
ADP	C00008	426.0219	0.65
AMP	C00020	346.0555	0.71
GTP	C00044	521.9827	0.62
GDP	C00035	442.0166	0.63
GMP	C00144	362.0505	0.68
Guanosine	C00387	282.0841	0.75
Cytidine	C00475	242.0791	0.72
dTMP	C00364	321.0498	0.71
Thymine	C00178	125.0343	0.71
UDP	C00015	402.9945	0.62
UMP	C00105	323.0283	0.67
Inosine	C00294	267.0731	0.75
Hypoxanthine	C00262	135.0300	0.73
Xanthine	C00385	151.0251	0.75
(S)(+)-Allantoin	C02350	157.0355	0.74
3',5'-Cyclic AMP	C00575	328.0451	0.76
5'-Phosphoribosyl-N-formylglycinamide	C04376	313.0441	0.67
5-6-Dihydrothymine	C00906	127.0499	0.73
Pyridoxal	C00250	166.0499	0.77
UDP-glucose	C00029	565.0473	0.63
Diphosphate	C00013	176.9349	0.65
D-Glucose	C00031	179.0552	0.72
D-Fructose 1-6-bisphosphate	C00354	338.9883	0.62
2/3-Phospho-D-glycerate	C00631	184.9846	0.63
Pyruvate	C00022	87.0073	0.70
Lactate	C01432	89.0229	0.69
Mannitol	C00392	181.0703	0.75
D-Arabitol	C01904	151.0604	0.73
Citrate	C00158	191.0188	0.66
2-Oxoglutarate	C00026	145.0131	0.63
Succinate	C00042	117.0179	0.65
Fumarate	C00122	115.0023	0.65
Malate	C00149	133.0129	0.64
2-Hydroxyglutarate/Citramalate	C02630	147.0288	0.65
6-Phospho-D-gluconate	C00345	275.0176	0.62
alpha-D-Ribose 1-phosphate	C00620	229.0114	0.67
5-Oxoproline	C01879	128.0340	0.69
S-Glutathionyl-L-cysteine	C05526	425.0806	0.67
Ascorbate	C00072	175.0239	0.59
Dehydroascorbate	C05422	173.0081	0.65
gamma-L-Glutamyl-D-alanine	C03738	217.0823	0.71
Dimethylglycine	C01026	102.0546	0.68
Argininosuccinate	C03406	289.1150	0.67
UDP-N-acetyl-D-glucosamine	C00043	606.0743	0.64
CMP-N-acetylneuraminate	C00128	613.1400	0.63
N-Acetylornithine	C00437	173.0922	0.74
trans-4-Hydroxy-L-proline	C01157	130.0497	0.75
Pantothenol	C00864	218.1028	0.75
5-6-Dihydrouracil	C00429	113.0343	0.71
L-Methionine S-oxide	C02989	164.0375	0.73
5-Hydroxyindoleacetate	C05635	190.0527	0.75
Indole	C00463	116.0492	1.80
quinolinic acid	C03722	166.0162	0.71
g-Oxalo-crotonate	C03453	157.0123	0.94
Glycerol 3-phosphate	C00093	171.0053	0.67
Ethanolamine phosphate	C00346	140.0106	0.72
N-Methylethanolamine phosphate	C01210	154.0264	0.70
sn-glycero-3-Phosphoethanolamine	C01233	214.0478	0.71
CDP-choline	C00307	487.0990	0.67
Pentanoate (valerate)	C00803	101.0594	1.80
Hexanoic acid (caproate)	C01585	115.0751	1.86
Cholesterol sulfate	C18043	465.3048	2.12
Glycochenodeoxycholic acid	C05466	448.3067	1.86
Tauroursodeoxycholic/taurochenodeoxycholic/taurodeoxycholic acid	BA109	498.2892	1.86
S-Acylglutathione	C02589	333.0590	0.67
Oxalosuccinate	C05379	189.0066	1.28
METABOLITES_END
#END