#METABOLOMICS WORKBENCH golgo8128_20231016_211635 DATATRACK_ID:4404 STUDY_ID:ST003267 ANALYSIS_ID:AN005352 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: Fused silica capillary tubing 50 um i.d. x 200 cm (cut to
CH:CHROMATOGRAPHY_SUMMARY        	100 cm for analysis), Manufacturer: Molex, Material number: 106815-0017, Product
CH:CHROMATOGRAPHY_SUMMARY        	description: TSP050375, Lot: CHCC04A
CH:INSTRUMENT_NAME               	Agilent 7100 CE
CH:COLUMN_NAME                   	Molex Fused silica capillary tubing (100cm x 50um)
CH:COLUMN_TEMPERATURE            	20
CH:FLOW_GRADIENT                 	(Not applicable)
CH:FLOW_RATE                     	10 uL/min
CH:INTERNAL_STANDARD             	Methionine sulfone
CH:SAMPLE_INJECTION              	Pressure injection 50 mbar, 5 sec (approximately 5 nL)
CH:SOLVENT_A                     	(Not applicable)
CH:SOLVENT_B                     	(Not applicable)
CH:CAPILLARY_VOLTAGE             	Positive, 30 kV
CH:RUNNING_BUFFER                	1 M formic acid
CH:SHEATH_LIQUID                 	50% (v/v) methanol-water containing 0.01 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 6230 TOF
MS:INSTRUMENT_TYPE               	TOF-MS
MS:MS_TYPE                       	ESI
MS:MS_COMMENTS                   	ESI-TOFMS was performed in positive ion mode. Automatic recalibration of each
MS:MS_COMMENTS                   	acquired spectrum was achieved using the masses of the reference standards (13C
MS:MS_COMMENTS                   	isotopic ion of a protonated methanol dimer, m/z 66.0631) and (protonated ion of
MS:MS_COMMENTS                   	hexakis(2,2-difluoroethoxy)phosphazene, m/z 622.0290). For system control and
MS:MS_COMMENTS                   	data acquisition we used the MassHunter workstation software for Agilent
MS:MS_COMMENTS                   	CE-TOFMS. CE-TOFMS raw data were analyzed using our proprietary software
MS:MS_COMMENTS                   	MasterHands (ver, 2.20.0.3).
MS:ION_MODE                      	POSITIVE
MS:CAPILLARY_VOLTAGE             	4,000 V
MS:DRY_GAS_FLOW                  	Nitrogen, 10 L/min
MS:DRY_GAS_TEMP                  	300℃
MS:FRAGMENT_VOLTAGE              	75 V
MS:IONIZATION                    	ESI
MS:OCTPOLE_VOLTAGE               	500 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	
1-Methylnicotinamide	0.6203	0.6566	0.6566	0.7383	0.8715	0.9424	0.9802	0.8910
2AB	0.0851	0.0668	0.0710	0.0817	0.1179	0.1288	0.0549	0.0862
5-Methylthioadenosine	0.0174	0.0202	0.0169	0.0176	0.0236	0.0304	0.0251	0.0258
Adenine	0.0250	0.0151	0.0144			0.0334		
Adenosine	0.0583	0.0708	0.0363	0.0240	0.0209	0.0225	0.0223	0.0174
Ala	2.0167	1.7485	1.8162	2.0305	2.2718	1.7902	1.7134	1.5391
alpha-Aminoadipate	0.0510	0.0657	0.0451	0.0730	0.0714	0.0994	0.1110	0.0865
Arg	0.6493	0.6755	0.5817	0.6412	0.4670	0.5142	0.4842	0.5047
Asn	0.3589	0.2758	0.4341	0.3141	0.3486	0.3228	0.2674	0.2517
Asp	1.3944	1.1461	1.1848	1.3279	4.2322	2.9359	3.0201	2.7742
beta-Ala	0.5740	0.4769	0.3390	0.6620	0.7618	1.0795	1.0536	1.0071
Betaine	0.6058	0.2380	0.3120	0.3022	1.0635	0.6904	0.6953	0.4107
Carnitine	0.2241	0.2126	0.2003	0.2461	0.2940	0.3580	0.3501	0.3258
Choline	0.5675	0.6322	0.3336	0.3396	0.3673	0.4513	0.7171	0.7701
Citrulline	0.0571	0.0538	0.0692	0.0732	0.0659	0.0738	0.0668	0.0577
Creatine	2.4851	2.2359	2.1353	2.9372	3.1658	3.8009	3.9274	3.5737
Creatinine	0.0395	0.0284	0.0303	0.0425	0.0589	0.0459	0.0457	0.0464
Cyclohexylamine			0.0301					0.0427
Cystathionine	0.2130	0.2086	0.2240	0.2703	0.2226	0.2841	0.2619	0.2834
Cysteine-glutathione disulphide		0.1219	0.2156					
Cytidine	0.0637	0.1052	0.0686	0.0492	0.0448	0.0447	0.0367	0.0364
Diethanolamine	1.3106	1.1094	1.6323	2.1281	2.1284	2.1442	3.0699	2.1246
GABA	0.0728	0.0339	0.0348	0.0279	0.1405	0.0934	0.0541	0.0471
gamma-Butyrobetaine	0.0306	0.0195	0.0179	0.0295	0.0375	0.0346	0.0355	0.0390
gamma-Glu-cys	0.1143	0.0684	0.0999	0.1130	0.1865	0.1669	0.2020	0.1980
Gln	12.6451	11.3369	11.1312	16.3872	11.8673	13.7947	14.2110	13.6111
Glu	41.8008	38.9709	40.4195	56.4068	53.7099	61.4313	61.8941	60.8364
Glutathione(ox)	5.9282	11.6172	7.2906	5.4072	6.7051	8.3875	3.9919	4.9802
Glutathione(red)	16.5984	6.3634	8.7573	26.0801	24.3821	37.8284	42.1659	42.9221
Gly	3.9676	3.0291	2.9869	3.6584	5.2795	4.4573	4.3740	3.7511
Glycerophosphorylcholine	0.6007	0.5643	0.8551	0.5418	2.0167	1.0633	1.2166	1.2436
Guanosine	0.0249	0.0311	0.0337	0.0243				
His	1.0199	0.9202	0.9690	1.1888	0.9190	0.9864	0.9605	1.0232
Hydroxyproline	0.1215	0.1033	0.1092	0.1348	0.1343	0.1485	0.1701	0.1320
Hypotaurine	0.3734	0.3316	0.2540	0.4675	0.4784	0.8030	0.7916	0.7637
Hypoxanthine	0.3531	0.4733	0.1688	0.1388	0.1082	0.1312	0.1826	0.1228
Ile	3.0714	2.8309	2.8646	3.8265	2.6648	3.1713	3.2884	3.0655
Inosine	0.2785	0.3487	0.1613	0.1013	0.0773	0.0793	0.0946	0.0697
Leu	3.2121	3.1744	2.9982	4.0469	2.9666	3.4129	3.6303	3.4198
Lys	1.3665	1.4010	1.4084	1.3668	0.9218	0.9569	0.9287	0.9355
Melamine	0.0618	0.1458	0.2435	0.1557	0.1203	0.0891	0.1202	0.1072
Met	0.8036	0.7540	0.7287	1.0303	0.6809	0.7743	0.8411	0.8042
Methionine sulfoxide	0.0584	0.0550		0.0623				
N1-Acetylspermidine	0.0236	0.0261	0.0199	0.0274	0.0084	0.0179	0.0163	0.0153
N6_N6_N6-Trimethyllysine	0.0267	0.0312	0.0290	0.0245	0.0127	0.0112	0.0152	0.0102
N8-Acetylspermidine	0.0054	0.0067		0.0077	0.0087	0.0093	0.0104	0.0095
N-Acetylglucosamine	0.3159	0.3051	0.2642	0.2727	0.4112	0.3375	0.4378	0.3337
N-Acetylputrescine				0.0119		0.0235		0.0213
Nicotinamide	0.0463	0.0399	0.0322	0.0583		0.0571	0.0528	0.0584
N_N-Dimethylglycine	0.0736	0.0237	0.0399	0.0626	0.1186	0.0653	0.0845	0.0669
o-Acetylcarnitine	0.0778	0.0643	0.0622	0.0766	0.0713	0.0873	0.0982	0.0749
Ornithine	0.1365	0.1120	0.1482	0.1147	0.2550	0.1761	0.1188	0.1450
Phe	1.9237	1.7951	1.7428	2.3839	1.5681	2.0085	1.9252	1.9119
Phosphorylcholine	6.3411	5.7788	4.9462	7.9044	8.1145	11.9092	12.2606	12.1060
Pro	0.9616	0.9464	0.9837	1.0991	1.6627	1.6731	1.7035	1.6324
Putrescine(1_4-Butanediamine)	0.0958	0.0967	0.0854	0.1118	0.0741	0.1000	0.1153	0.0982
Pyridoxine	0.0254	0.0247	0.0251	0.0226	0.0344	0.0327	0.0358	0.0290
SAH	0.0164	0.0250	0.0150	0.0231		0.0299		0.0204
SAM+	0.1147	0.1264	0.1125	0.1267	0.1280	0.1727	0.1600	0.1630
Ser	2.0771	1.7643	1.7773	2.2382	2.5538	2.4426	2.2373	2.0353
S-Lactoylglutathione		0.1458	0.0330	0.0820	0.0490		0.0646	0.0499
Spermidine	0.0547	0.0389	0.0426	0.0685	0.0289	0.0442	0.0625	0.0412
Taurine	2.6430	2.4766	1.6384	3.1846	4.0395	5.7997	6.3081	4.9694
Thiamine	0.0798	0.0803	0.0830	0.0941	0.0886	0.0969	0.1011	0.0872
Thr	5.3770	5.1535	5.2086	6.6747	5.0283	5.4647	5.2345	5.3186
Thymidine	1.4497	1.3396	1.6493	1.4119	2.5669	2.3690	2.2927	2.0509
Trimethylamine N-oxide	0.0370	0.0366	0.0254	0.0252	0.0463	0.0513	0.0515	0.0367
Trp	0.5657	0.5467	0.5370	0.7396	0.5025	0.5432	0.5356	0.5528
Tyr	2.6578	2.5122	2.4477	3.2156	2.1598	2.5663	2.4955	2.5683
Urea	6.2008	24.9999	7.5221	6.3525	9.4393	5.1033	3.7700	19.7347
Uridine	1.4958	1.0164	1.0561	1.1681	2.2930	2.1907	2.0881	1.7882
Val	3.4305	3.3920	3.3522	4.1805	3.0074	3.5751	3.6052	3.5125
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	
1-Methylnicotinamide			C02918						
2AB			C02356						
5-Methylthioadenosine			C00170						
Adenine			C00147						
Adenosine			C00212						
Ala			C00041						
alpha-Aminoadipate			C00956						
Arg			C00062						
Asn			C00152						
Asp			C00049						
beta-Ala			C00099						
Betaine			C00719						
Carnitine			C00318						
Choline			C00114						
Citrulline			C00327						
Creatine			C00300						
Creatinine			C00791						
Cyclohexylamine			C00571						
Cystathionine			C02291						
Cysteine-glutathione disulphide			-						
Cytidine			C00475						
Diethanolamine			C06772						
GABA			C00334						
gamma-Butyrobetaine			C01181						
gamma-Glu-cys			C00669						
Gln			C00064						
Glu			C00025						
Glutathione(ox)			C00127						
Glutathione(red)			C00051						
Gly			C00037						
Glycerophosphorylcholine			C00670						
Guanosine			C00387						
His			C00135						
Hydroxyproline			C01157						
Hypotaurine			C00519						
Hypoxanthine			C00262						
Ile			C00407						
Inosine			C00294						
Leu			C00123						
Lys			C00047						
Melamine			C08737						
Met			C00073						
Methionine sulfoxide			C02989						
N1-Acetylspermidine			C00612						
N6,N6,N6-Trimethyllysine			C03793						
N8-Acetylspermidine			C01029						
N-Acetylglucosamine			C00140						
N-Acetylputrescine			C02714						
Nicotinamide			C00153						
N,N-Dimethylglycine			C01026						
o-Acetylcarnitine			C02571						
Ornithine			C00077						
Phe			C00079						
Phosphorylcholine			C00588						
Pro			C00148						
Putrescine(1,4-Butanediamine)			C00134						
Pyridoxine			C00314						
SAH			C00021						
SAM+			C00019						
Ser			C00065						
S-Lactoylglutathione			C03451						
Spermidine			C00315						
Taurine			C00245						
Thiamine			C00378						
Thr			C00188						
Thymidine			C00214						
Trimethylamine N-oxide			C01104						
Trp			C00078						
Tyr			C00082						
Urea			C00086						
Uridine			C00299						
Val			C00183						
METABOLITES_END
#END