#METABOLOMICS WORKBENCH golgo8128_20231016_211635 DATATRACK_ID:4404 STUDY_ID:ST003267 ANALYSIS_ID:AN005353 PROJECT_ID:PR002029
VERSION                          	1
CREATED_ON                       	08-21-2024
#PROJECT
PR:PROJECT_TITLE                 	FDX2-KO induces global down-regulation of iron-sulfur cluster-containing
PR:PROJECT_TITLE                 	proteins and senescence-like growth arrest or death in ovarian cancer cells
PR:PROJECT_SUMMARY               	Recent studies report molecular mechanisms underlying iron-sulfur cluster (Fe-S)
PR:PROJECT_SUMMARY               	biosynthesis and suggest its importance in health and disease. However, a role
PR:PROJECT_SUMMARY               	for Fe-S biosynthesis in cancer contexts remains unclear. Here we report that
PR:PROJECT_SUMMARY               	FDX2, an Fe-S assembly factor, is indispensable for maintenance of cellular
PR:PROJECT_SUMMARY               	Fe-S-containing proteins (Fe-S protein(s)) and proliferation of ovarian cancer
PR:PROJECT_SUMMARY               	(OVC) cells. CRISPR-screening of all metabolism-related genes in OVC cells
PR:PROJECT_SUMMARY               	identified several Fe-S assembly genes as essential for OVC growth. Using an
PR:PROJECT_SUMMARY               	inducible FDX2-KO OVC line, we found that FDX2 loss promotes either
PR:PROJECT_SUMMARY               	senescence-like growth arrest or cell death, depending on TP53 status.
PR:PROJECT_SUMMARY               	Mechanistically, FDX2-loss caused global but differential post transcriptional
PR:PROJECT_SUMMARY               	down-regulation of Fe-S proteins, in turn perturbing respiration,
PR:PROJECT_SUMMARY               	iron-regulation and redox homeostasis, all associated with DNA damage. These
PR:PROJECT_SUMMARY               	results demonstrate significant roles for Fe-S biosynthesis in OVC proliferation
PR:PROJECT_SUMMARY               	and survival and provide information about how the cellular Fe-S-protein network
PR:PROJECT_SUMMARY               	responds to disruptions in Fe-S assembly.
PR:INSTITUTE                     	Tohoku University
PR:DEPARTMENT                    	Graduate School of Medicine
PR:LABORATORY                    	Department of Biochemical Oncology
PR:LAST_NAME                     	Tanuma
PR:FIRST_NAME                    	Nobu-hiro
PR:ADDRESS                       	2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan
PR:EMAIL                         	nobuhiro.tanuma.c7@tohoku.ac.jp
PR:PHONE                         	+81-22-381-1165
PR:PUBLICATIONS                  	https://doi.org/10.1016/j.jbc.2024.107678,
PR:PUBLICATIONS                  	https://www.jbc.org/article/S0021-9258(24)02179-3/fulltext,
PR:PUBLICATIONS                  	https://pubmed.ncbi.nlm.nih.gov/39151727/
PR:DOI                           	http://dx.doi.org/10.21228/M8BJ9X
#STUDY
ST:STUDY_TITLE                   	FDX2-KO induces global down-regulation of iron-sulfur cluster-containing
ST:STUDY_TITLE                   	proteins and senescence-like growth arrest or death in ovarian cancer cells
ST:STUDY_SUMMARY                 	Recent studies report molecular mechanisms underlying iron-sulfur cluster (Fe-S)
ST:STUDY_SUMMARY                 	biosynthesis and suggest its importance in health and disease. However, a role
ST:STUDY_SUMMARY                 	for Fe-S biosynthesis in cancer contexts remains unclear. Here we report that
ST:STUDY_SUMMARY                 	FDX2, an Fe-S assembly factor, is indispensable for maintenance of cellular
ST:STUDY_SUMMARY                 	Fe-S-containing proteins (Fe-S protein(s)) and proliferation of ovarian cancer
ST:STUDY_SUMMARY                 	(OVC) cells. CRISPR-screening of all metabolism-related genes in OVC cells
ST:STUDY_SUMMARY                 	identified several Fe-S assembly genes as essential for OVC growth. Using an
ST:STUDY_SUMMARY                 	inducible FDX2-KO OVC line, we found that FDX2 loss promotes either
ST:STUDY_SUMMARY                 	senescence-like growth arrest or cell death, depending on TP53 status.
ST:STUDY_SUMMARY                 	Mechanistically, FDX2-loss caused global but differential post transcriptional
ST:STUDY_SUMMARY                 	down-regulation of Fe-S proteins, in turn perturbing respiration,
ST:STUDY_SUMMARY                 	iron-regulation and redox homeostasis, all associated with DNA damage. These
ST:STUDY_SUMMARY                 	results demonstrate significant roles for Fe-S biosynthesis in OVC proliferation
ST:STUDY_SUMMARY                 	and survival and provide information about how the cellular Fe-S-protein network
ST:STUDY_SUMMARY                 	responds to disruptions in Fe-S assembly.
ST:INSTITUTE                     	Tohoku University
ST:DEPARTMENT                    	Graduate School of Medicine
ST:LABORATORY                    	Department of Biochemical Oncology
ST:LAST_NAME                     	Tanuma
ST:FIRST_NAME                    	Nobu-hiro
ST:ADDRESS                       	2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan
ST:EMAIL                         	nobuhiro.tanuma.c7@tohoku.ac.jp
ST:PHONE                         	+81-22-381-1165
ST:SUBMIT_DATE                   	2023-10-16
#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]Additional sample data
SUBJECT_SAMPLE_FACTORS           	-	5	Sample source:Ovarian cancer cells | DOX:OFF	Amount of protein (ug)=617.1; Day=Day 5; RAW_FILE_NAME=5_d1
SUBJECT_SAMPLE_FACTORS           	-	6	Sample source:Ovarian cancer cells | DOX:OFF	Amount of protein (ug)=701.8; Day=Day 5; RAW_FILE_NAME=6_d1
SUBJECT_SAMPLE_FACTORS           	-	7	Sample source:Ovarian cancer cells | DOX:OFF	Amount of protein (ug)=726; Day=Day 5; RAW_FILE_NAME=7_d1
SUBJECT_SAMPLE_FACTORS           	-	8	Sample source:Ovarian cancer cells | DOX:OFF	Amount of protein (ug)=713.9; Day=Day 5; RAW_FILE_NAME=8_d1
SUBJECT_SAMPLE_FACTORS           	-	1	Sample source:Ovarian cancer cells | DOX:On	Amount of protein (ug)=399.3; Day=Day 5; RAW_FILE_NAME=1_d1
SUBJECT_SAMPLE_FACTORS           	-	2	Sample source:Ovarian cancer cells | DOX:On	Amount of protein (ug)=435.6; Day=Day 5; RAW_FILE_NAME=2_d1
SUBJECT_SAMPLE_FACTORS           	-	3	Sample source:Ovarian cancer cells | DOX:On	Amount of protein (ug)=459.8; Day=Day 5; RAW_FILE_NAME=3_d1
SUBJECT_SAMPLE_FACTORS           	-	4	Sample source:Ovarian cancer cells | DOX:On	Amount of protein (ug)=435.6; Day=Day 5; RAW_FILE_NAME=4_d1
#COLLECTION
CO:COLLECTION_SUMMARY            	Cells were maintained in DMEM medium (high glucose) in the presence of
CO:COLLECTION_SUMMARY            	doxycycline (DOX). For metabolome analysis, cells were seeded to new dishes at
CO:COLLECTION_SUMMARY            	day 0 and cultured in the presence or absence of Dox for 4 days. At day 4, cells
CO:COLLECTION_SUMMARY            	were replenished with fresh medium (with or without Dox) and cultured for
CO:COLLECTION_SUMMARY            	additional one day. At day 5, cells were washed with PBS twice and detached from
CO:COLLECTION_SUMMARY            	dishes using cell scraper into PBS. The detached cells were collected by
CO:COLLECTION_SUMMARY            	centrifugation, snap-frozen by liquid nitrogen, and stored at -80 oC until
CO:COLLECTION_SUMMARY            	metabolite extraction.
CO:SAMPLE_TYPE                   	Cultured cells
#TREATMENT
TR:TREATMENT_SUMMARY             	FDX2-iKO JHOC5 cells were cultured 5 days with or without doxycycline (30 ng/ml)
TR:TREATMENT_SUMMARY             	and collected. Metabolome data were normalized to protein amounts of cells
TR:TREATMENT_SUMMARY             	(shown in "Study design") used for the metabolite extraction.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	The cell extracts were centrifugally concentrated for 2.5 hours using a LABCONCO
SP:SAMPLEPREP_SUMMARY            	centrifugal concentrator with cooling function (LABCONCO corporation).
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Column information: COSMO(+)Capillary, 50 um i.d. x 120 cm (cut to 105 cm for
CH:CHROMATOGRAPHY_SUMMARY        	analysis), Manufacturer: Nacalai Tesque，Product No: 07584-44，Lot: P23753
CH:INSTRUMENT_NAME               	Agilent 7100 CE
CH:COLUMN_NAME                   	Nacalai Tesque COSMO(+)Capillary (105cm x 50um)
CH:COLUMN_TEMPERATURE            	20
CH:FLOW_GRADIENT                 	(Not applicable)
CH:FLOW_RATE                     	10 uL / min
CH:INTERNAL_STANDARD             	CAS
CH:SAMPLE_INJECTION              	Pressure injection 50 mbar, 30 sec (approximately 30 nL)
CH:SOLVENT_A                     	(Not applicable)
CH:SOLVENT_B                     	(Not applicable)
CH:CAPILLARY_VOLTAGE             	Negative, 30 kV
CH:RUNNING_BUFFER                	50 mM ammonium acetate, pH 8.5
CH:SHEATH_LIQUID                 	5 mM ammonium acetate in 50% (v/v) methanol-water containing 0.1 uM
CH:SHEATH_LIQUID                 	hexakis(2,2-difluoroethoxy)phosphazene
CH:CHROMATOGRAPHY_TYPE           	CE
#ANALYSIS
AN:LABORATORY_NAME               	Institute for Advanced Biosciences, Keio University
AN:ANALYSIS_TYPE                 	MS
AN:ACQUISITION_DATE              	April 3, 2024
AN:SOFTWARE_VERSION              	MassHunter workstation software version B.08.00
#MS
MS:INSTRUMENT_NAME               	Agilent 6210 TOF
MS:INSTRUMENT_TYPE               	TOF-MS
MS:MS_TYPE                       	ESI
MS:MS_COMMENTS                   	ESI-TOFMS was performed in negative ion mode. Automatic recalibration of each
MS:MS_COMMENTS                   	acquired spectrum was performed using reference masses of standards, i.e., (13C
MS:MS_COMMENTS                   	isotopic ion of deprotonated acetatic acid dimer, m/z 120.0383) and
MS:MS_COMMENTS                   	([hexakis(2,2-difluoroethoxy)phosphazene + deprotonated acetic acid, m/z
MS:MS_COMMENTS                   	680.0355). For system control and data acquisition we used the MassHunter
MS:MS_COMMENTS                   	workstation software for Agilent CE-TOFMS. CE-TOFMS raw data were analyzed using
MS:MS_COMMENTS                   	our proprietary software MasterHands (ver, 2.20.0.3).
MS:ION_MODE                      	NEGATIVE
MS:CAPILLARY_VOLTAGE             	3,500 V
MS:DRY_GAS_FLOW                  	Nitrogen, 10 L/min
MS:DRY_GAS_TEMP                  	300℃
MS:FRAGMENT_VOLTAGE              	100 V
MS:IONIZATION                    	ESI
MS:OCTPOLE_VOLTAGE               	200 V
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS         	μmol/g-protein
MS_METABOLITE_DATA_START
Samples	5	6	7	8	1	2	3	4
Factors	Sample source:Ovarian cancer cells | DOX:OFF	Sample source:Ovarian cancer cells | DOX:OFF	Sample source:Ovarian cancer cells | DOX:OFF	Sample source:Ovarian cancer cells | DOX:OFF	Sample source:Ovarian cancer cells | DOX:On	Sample source:Ovarian cancer cells | DOX:On	Sample source:Ovarian cancer cells | DOX:On	Sample source:Ovarian cancer cells | DOX:On	
2-Hydroxyglutarate		0.0561		0.0464	0.0680	0.0497	0.0585	0.0643
2-Oxoglutarate	0.4600	0.3522	0.3401	0.4312	0.4495	0.4054	0.3860	0.5304
2PG							0.2934	0.2705
3PG	1.5929	1.7476	1.0931	1.2851	1.4640	1.6840	1.7802	1.9295
3-Phenylpropionate	0.3291	0.3223	0.2986	0.3066	0.5951	0.5346	0.6174	0.5921
4-Acetylbutyrate		0.0971	0.0993	0.0950	0.1889	0.1288	0.1555	
4-Oxopentanoate	0.2293	0.0789	0.1567	0.1512		0.2895	0.2546	0.3085
5-Oxoproline	1.0813	1.2855	0.9113	0.8389	1.5006	1.0835	1.3012	1.3519
6-Phosphogluconate	0.2261	0.2826	0.2849	0.2164	0.3150	0.3050	0.2474	0.1949
Acetyl CoA		0.0576	0.0516	0.0673	0.0375		0.0474	
ADP	2.6909	2.6104	2.5952	2.1689	2.6972	2.7021	2.2845	2.3210
AMP	1.4141	1.6946	0.8742	0.5780	0.6512	0.4730	0.5085	0.3174
ATP	2.1361	2.1129	3.0065	4.6272	4.6520	7.9097	5.7462	7.3988
Azelate	0.0712	0.0569	0.0588	0.0624	0.1437	0.0813	0.1013	0.0743
Benzoate	0.9465	0.8311	1.2272	1.0041	0.8229	1.5031	1.1371	1.3017
CDP	0.2410	0.2433	0.1911	0.2092	0.1819	0.2158	0.1463	0.0938
cis-Aconitate	0.0730	0.0665	0.0481	0.0230		0.0766	0.0290	
Citrate	1.2872	1.1183	1.1149	1.0555	0.9285	1.0732	0.9507	0.9599
CMP	0.2100	0.1987	0.1303	0.0890				
CoA		0.0388				0.0368		
CTP	0.2642	0.2416	0.3858	0.5666	0.5789	0.8854	0.6940	0.8371
Decanoate	0.0669	0.0642	0.0625		0.0972	0.0705	0.0926	0.0696
DHAP	3.6763	3.6748	2.2091	2.1553	1.8515	2.5775	2.9461	2.7271
Dodecanoate	0.1801	0.1995	0.1194	0.1437	0.2626	0.2478	0.2744	0.3076
Ethanolamine phosphate	0.5841	0.4899	0.4611	0.6139	0.9795	0.7133	0.6375	0.7087
F1_6P	5.4721	6.1513	5.9953	4.8925	5.1684	5.9325	4.9644	3.5787
F6P	0.1056	0.0682	0.1399					
Fumarate	0.4209	0.3954	0.2927	0.4105	0.5836	0.5133	0.7569	0.7281
G1P	0.3711	0.3799	0.3472	0.3165	0.2513	0.3023	0.3499	0.2245
G3P	0.3757	0.2905			0.2896			
G6P	0.4636	0.6195	0.7725	0.3921	0.7046	0.3483	0.3193	0.1655
GDP	0.6338	0.5669	0.5471	0.5035	0.6213	0.6305	0.5106	0.4576
Gluconate	0.3774	0.2767	0.2768	0.4951	0.5691	0.8290	0.7496	0.7093
Glycerophosphate	0.3503	0.3964	0.2182	0.3614	0.2567	0.2464	0.2797	0.2626
Glycolate	1.4803	1.2782	1.3602	1.1947	2.9781	2.1515	1.2081	1.7543
GMP	0.1528	0.2128	0.0930	0.0688	0.0945	0.1419		
GTP	0.2413	0.3113	0.3860	0.6182	0.5445	1.1256	0.6787	0.9936
Heptanoate	0.0409							0.0443
Lactate	18.3171	17.3658	15.2625	18.1600	31.5587	16.7197	16.8457	19.4503
Malate	1.9357	1.8707	1.6296	1.8117	3.3857	2.8574	2.8480	3.1859
Malonate	0.1721	0.1200	0.1069	0.1374	0.2097	0.1670		0.2142
N-Acetylaspartate	0.1349	0.1056	0.0924	0.1350	0.1778	0.1677	0.1663	0.1357
N-Acetylglucosamine 1-phosphate		0.0644						
N-Acetylglutamate			0.0454	0.0335			0.0473	
NAD+	0.7178	0.6058	0.7152	0.8193	0.9586	0.8908	0.7545	0.8540
NADH	0.4379	0.6523	0.4387	0.5682	0.2869	0.5711	0.7693	0.5645
NADPH			0.0784			0.1313		
Pantothenate	0.5453	0.4357	0.4726	0.7292	0.9302	1.0220	1.0143	0.9549
Pelargonate	0.4027	0.4002	0.3778	0.2404	0.5882	0.4912	0.4780	0.3529
PEP		0.0895						
Phthalate	0.0200	0.0150	0.0255	0.0179	0.0404	0.0366	0.0354	0.0388
R5P	0.2121	0.1990	0.1255	0.1191			0.1885	0.1369
Ru5P	0.9678	1.0985	0.5101	0.4685	0.3413	0.3900	0.4492	0.5595
Sebacate	0.0171	0.0084			0.0223		0.0188	
Succinate	2.0522	1.9499	1.4564	1.3598	1.2383	1.1693	1.3212	1.2473
Terephthalate	0.1652	0.1235	0.1055	0.1598	0.2522	0.2183	0.2021	0.2707
Threonate	0.0938	0.1226		0.1296	0.3106			
UDP	0.6511	0.5711	0.4711	0.5144	0.5270	0.4382	0.3829	0.4515
UDP-glucose	2.1147	2.2187	1.9474	2.2354	1.8525	2.1397	2.2826	2.0418
UDP-glucuronate	0.2827	0.3048	0.3230	0.2678	0.2486	0.3897	0.3511	0.3337
UDP-N-acetylglucosamine	3.2386	3.4831	2.9852	2.9829	3.1471	3.0685	3.4228	2.9352
UMP	0.6984	0.7318	0.2935	0.2914	0.2489	0.2528	0.1933	0.1291
UTP	0.7087	0.6196	0.8448	1.3877	1.2771	2.1039	1.7153	1.9943
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	pubchem_id	inchi_key	kegg_id	other_id	other_id_type	ri	ri_type	moverz_quant	
2-Hydroxyglutarate			C02630						
2-Oxoglutarate			C00026						
2PG			C00631						
3PG			C00197						
3-Phenylpropionate			C05629						
4-Acetylbutyrate			C02129						
4-Oxopentanoate			-						
5-Oxoproline			C01879						
6-Phosphogluconate			C00345						
Acetyl CoA			C00024						
ADP			C00008						
AMP			C00020						
ATP			C00002						
Azelate			C08261						
Benzoate			C00180						
CDP			C00112						
cis-Aconitate			C00417						
Citrate			C00158						
CMP			C00055						
CoA			C00010						
CTP			C00063						
Decanoate			C01571						
DHAP			C00111						
Dodecanoate			C02679						
Ethanolamine phosphate			C00346						
F1,6P			C00354						
F6P			C00085						
Fumarate			C00122						
G1P			C00103						
G3P			C00661						
G6P			C00092						
GDP			C00035						
Gluconate			C00257						
Glycerophosphate			C00093						
Glycolate			C00160						
GMP			C00144						
GTP			C00044						
Heptanoate			C17714						
Lactate			C00186						
Malate			C00711						
Malonate			C00383						
N-Acetylaspartate			C01042						
N-Acetylglucosamine 1-phosphate			C04501						
N-Acetylglutamate			C00624						
NAD+			C00003						
NADH			C00004						
NADPH			C00005						
Pantothenate			C00864						
Pelargonate			C01601						
PEP			C00074						
Phthalate			C01606						
R5P			C00117						
Ru5P			C00199						
Sebacate			C08277						
Succinate			C00042						
Terephthalate			C06337						
Threonate			C01620						
UDP			C00015						
UDP-glucose			C00029						
UDP-glucuronate			C00167						
UDP-N-acetylglucosamine			C00043						
UMP			C00105						
UTP			C00075						
METABOLITES_END
#END