#METABOLOMICS WORKBENCH RCulpHill_20210318_135702_mwtab.txt DATATRACK_ID:2535 STUDY_ID:ST001731 ANALYSIS_ID:AN002817 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        	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	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
L-alanine	25846710	27350690	22640420	19739830	32446750	19241770	28228450	28853130
L-arginine	33069100	29532740	32699410	34615490	29849810	22246150	26777620	29345380
L-asparagine	5287412	5712984	5482410	4585582	6469310	5051520	5509352	5743908
L-aspartate	7744486	9767284	8514543	5942422	11743320	9192721	11284300	10571520
L-cysteine	1760250	1921405	2544354	2048566	2161260	1869524	2426912	2554490
L-glutamate	118737400	115313100	103363900	95843420	151418100	102915700	123467200	128029200
L-glutamine	95674020	101819200	102486500	71178670	132242000	85573050	115139300	110588900
glycine	8511617	7847110	7520590	6644040	6981228	6922066	9191731	8462046
L-histidine	10195650	12795450	11361920	11110960	11841090	9188629	11684340	11789300
L-leucine	253702300	273581800	285953400	242578500	310626700	219719600	286417700	287651400
L-lysine	10238570	10186600	11436220	11038280	10564990	9040827	11533560	10673670
L-methionine	26931780	29639050	29281590	26628580	34055060	23554140	31454610	32121870
L-phenylalanine	31094620	42876280	38289340	36309640	44042860	31238240	43555600	40351850
L-proline	376454400	404296200	392707700	296764900	437001200	355542000	459411000	437958600
L-serine	2761064	3115084	2715200	2266976	3268343	2518774	3051352	3138984
L-threonine	7078662	7688394	6838836	5719390	8483821	5525374	7649098	7105282
L-tryptophan	22457620	23074490	23190210	20156750	30479700	23686550	27599750	26350720
L-tyrosine	34303300	44180310	32633180	35617530	39521890	29973860	37357670	38929500
L-valine	80137990	93697440	94559420	70457500	92323840	70728160	91989880	89335260
Adenosine	5904584	6238426	7516610	9179527	6041276	7767116	6488536	6100758
Adenine	11725050	14417000	10863840	11631650	10541880	9417337	11089910	11568840
CMP	2905915	2653824	2322369	1684017	3132887	2433419	1849567	2205717
Cytosine	14003400	18059840	17592360	12259710	14349860	11975520	16052290	17317450
Uracil	1471416	1819037	1917365	1930754	1860153	1908022	1949554	1530906
5-Hydroxyisourate	17971210	16622690	16513140	13635450	16036350	13259540	18585030	17562470
Pyridoxamine 5'-phosphate	138242	145369	122009	132122	178237	137873	164350	154064
Nicotinamide	25937740	33106590	27544540	23228300	30222390	24493470	33707960	30177350
Adenylosuccinic acid	3829878	4757606	3196725	3952142	5286928	3413896	5512610	5151098
NAD+	6880292	6078630	6381926	5281322	6472016	6538806	7197498	5488570
Glutathione	760612600	959818000	670564600	758011600	824550500	650998600	737081900	800960300
Glutathione disulfide	815848	781441	618296	598322	861403	484255	762358	1053040
Cys-Gly	24666000	31923150	21413170	24131660	25782710	20590060	22283650	24843830
gamma-L-Glutamyl-L-cysteine	1936309	2546376	2030218	2503383	1990772	1594268	2397546	2324248
(5-L-Glutamyl)-L-glutamine	416624	493020	466722	333030	599162	253257	515357	334652
Cystathionine	942649	903867	851454	615778	908645	877311	889176	754997
3-Phosphonooxypyruvate	27127990	27184010	23606720	23392590	21564710	25239950	18761620	20507460
S-Adenosyl-L-methionine	692003	757538	744540	636278	684488	573117	772475	769889
Ornithine	1298919	1060553	1318459	1288522	1175074	1009997	1449459	1321369
L-Citrulline	12440860	13082960	12070680	9681652	21515620	16318370	18324060	17994410
Putrescine	528103	357380	753400	766658	626712	644544	744464	767300
Spermidine	2915288	3962484	3289903	4230644	3773698	1922031	3223454	3119205
Spermine	180763	947736	204805	240490	267204	118039	303550	218383
Carnosine	82335	111133	100412	52699	88041	75275	72296	72495
Creatine	189815900	194416100	183538900	159380100	192612800	169665100	221974300	222498700
Creatinine	4832528	5510490	4990990	3294872	5995050	4065866	5068952	5309172
Taurine	8202516	7056274	7981310	8140176	8742731	9833010	7389214	8911441
3-Methyleneoxindole	1734644	1800383	1682389	1516469	2063813	1588081	1784523	1906520
Indole-3-acetate	739588	751261	680865	635162	691647	650177	989291	907463
Anthranilate	1980056	2025308	1786506	1604287	2205746	1696813	2112770	2175124
Sphingosine	842395	912886	1107183	1005646	606683	514551	408756	351506
Acetylcholine	16190600	18041190	17931440	16426200	15792340	13694750	18214600	17368200
Choline	123368900	134727000	132083400	113105400	153814300	134558000	142865600	152758400
L-Carnitine	79395460	83849110	83522070	68760390	92450900	69952640	72722940	83649530
acyl-C2 (acetyl-carnitine)	109588700	103517800	102143800	93508900	99852220	81195380	97768680	106064600
acyl-C3 (propionyl-carnitine)	21114110	22144820	18486670	18261500	20715170	18561680	18512990	20563550
acyl-C4 (butanoyl-l-carnitine)	62208400	68381250	46233880	52246100	63442880	51312590	63310510	59930700
acyl-C5:1 (Tiglylcarnitine)	151634	177579	165184	179550	149926	152966	159774	148854
acyl-C5 (isovalerylcarnitine)	11720950	12809790	9975505	10729510	14681610	11280690	14066550	10838310
acyl-C4-OH (Hydroxybutyrylcarnitine)	1931822	2018188	1851870	1763097	2032013	1814673	1849993	2209741
acyl-C6 (hexanoyl-l-carnitine)	6995134	4835418	5417716	4922868	5961110	4702824	6327090	6369726
acyl-C8:1 (octenoyl-l-carnitine)	164784	158210	137889	163891	160271	157629	162626	166185
acyl-C8 (L-octanoylcarnitine	1167539	968766	705340	735740	1197937	865064	811476	794787
acyl-C10 (O-Decanoyl-L-carnitine)	239123	198353	140148	168727	233531	178351	175212	174459
acyl-C12:1 (O-dodecenoyl-carnitine)	129533	2855751	131729	155158	258158	167319	205586	2206665
cis-p-Coumarate	12987740	16333310	10542950	12656760	13583370	10491690	13073870	14271290
Shikimate 3-phosphate	230312	199866	196854	213839	276175	227788	234087	248630
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	CmpdID	parent	medRt
L-alanine	C00041	90.0555	0.66
L-arginine	C00062	175.1187	0.62
L-asparagine	C00152	133.0606	0.65
L-aspartate	C00049	134.0448	0.66
L-cysteine	C00097	122.0273	0.70
L-glutamate	C00025	148.0602	0.66
L-glutamine	C00064	147.0762	0.66
glycine	C00037	76.0400	1.81
L-histidine	C00135	156.0766	0.62
L-leucine	C00123	132.1019	0.69
L-lysine	C00047	147.1126	0.61
L-methionine	C00073	150.0579	0.68
L-phenylalanine	C00079	166.0860	0.70
L-proline	C00148	116.0708	0.68
L-serine	C00065	106.0502	0.65
L-threonine	C00188	120.0657	0.66
L-tryptophan	C00078	205.0968	1.73
L-tyrosine	C00082	182.0809	0.70
L-valine	C00183	118.0865	0.68
Adenosine	C00212	268.1032	0.69
Adenine	C00147	136.0617	0.71
CMP	C00055	324.0585	0.67
Cytosine	C00380	112.0508	0.70
Uracil	C00106	113.0348	0.68
5-Hydroxyisourate	C11821	185.0311	0.68
Pyridoxamine 5'-phosphate	C00647	249.0607	1.73
Nicotinamide	C00153	123.0554	0.69
Adenylosuccinic acid	C03794	464.0804	1.03
NAD+	C00003	664.1150	0.67
Glutathione	C00051	308.0902	0.68
Glutathione disulfide	C00127	613.1572	0.69
Cys-Gly	C01419	179.0482	0.68
gamma-L-Glutamyl-L-cysteine	C00669	251.0690	0.67
(5-L-Glutamyl)-L-glutamine	C05283	276.1185	0.67
Cystathionine	C00542	223.0744	0.65
3-Phosphonooxypyruvate	C03232	184.9853	3.80
S-Adenosyl-L-methionine	C00019	399.1435	0.63
Ornithine	C01602	133.0974	0.61
L-Citrulline	C00327	176.1027	0.66
Putrescine	C00134	89.1079	0.61
Spermidine	C00315	146.1651	0.61
Spermine	C00750	203.2226	0.61
Carnosine	C00386	227.1141	0.61
Creatine	C00300	132.0768	0.67
Creatinine	C00791	114.0665	0.67
Taurine	C00245	126.0221	0.66
3-Methyleneoxindole	C02796	146.0600	1.73
Indole-3-acetate	C00954	176.0705	1.80
Anthranilate	C00108	138.0549	0.93
Sphingosine	C00319	300.2888	3.79
Acetylcholine	C01996	146.1174	0.68
Choline	C00114	104.1073	0.66
L-Carnitine	C00318	162.1122	0.66
acyl-C2 (acetyl-carnitine)	C02571	204.1227	0.69
acyl-C3 (propionyl-carnitine)	C03017	218.1382	0.69
acyl-C4 (butanoyl-l-carnitine)	C02862	232.1538	0.70
acyl-C5:1 (Tiglylcarnitine)	HMDB02366	244.1539	1.75
acyl-C5 (isovalerylcarnitine)	HMDB00688	246.1694	1.75
acyl-C4-OH (Hydroxybutyrylcarnitine)	HMDB13127	248.1487	0.68
acyl-C6 (hexanoyl-l-carnitine)	HMDB00756	260.1851	1.80
acyl-C8:1 (octenoyl-l-carnitine)	ac111	286.2005	1.84
acyl-C8 (L-octanoylcarnitine	HMDB00791	288.2161	1.87
acyl-C10 (O-Decanoyl-L-carnitine)	C03299	316.2475	1.99
acyl-C12:1 (O-dodecenoyl-carnitine)	HMDB13326	342.2630	1.83
cis-p-Coumarate	C06738	165.0545	0.70
Shikimate 3-phosphate	C03175	255.0277	0.80
METABOLITES_END
#END