#METABOLOMICS WORKBENCH weishengxin_20240320_013216 DATATRACK_ID:4730 STUDY_ID:ST003134 ANALYSIS_ID:AN005144 PROJECT_ID:PR001947
VERSION             	1
CREATED_ON             	March 20, 2024, 9:12 pm
#PROJECT
PR:PROJECT_TITLE                 	Targeting SOX13 inhibits the assembly of respiratory chain supercomplexes to
PR:PROJECT_TITLE                 	overcome ferroptosis-resistance in gastric cancer
PR:PROJECT_TYPE                  	Untargeted metabolomic analysis
PR:PROJECT_SUMMARY               	Therapeutic resistance represents a bottleneck to treatment in advanced gastric
PR:PROJECT_SUMMARY               	cancer (GC). Ferroptosis is an iron-dependent form of non-apoptotic cell death
PR:PROJECT_SUMMARY               	and is associated with anti-cancer therapeutic efficacy. Further investigations
PR:PROJECT_SUMMARY               	are required to clarify the underlying mechanisms. Ferroptosis-resistant GC cell
PR:PROJECT_SUMMARY               	lines are constructed. Dysregulated mRNAs between ferroptosis-resistant and
PR:PROJECT_SUMMARY               	parental cell lines are identified. The expression of SOX13/SCAF1 is manipulated
PR:PROJECT_SUMMARY               	in GC cell lines where relevant biological and molecular analyses are performed.
PR:PROJECT_SUMMARY               	Molecular docking and computational screening are performed to screen potential
PR:PROJECT_SUMMARY               	inhibitors of SOX13. We show that SOX13 boosts protein remodeling of electron
PR:PROJECT_SUMMARY               	transport chain (ETC) complexes by directly transactivating SCAF1. This leads to
PR:PROJECT_SUMMARY               	increased supercomplexes (SCs) assembly, mitochondrial respiration,
PR:PROJECT_SUMMARY               	mitochondrial energetics and chemo- and immune-resistance. Zanamivir, reverts
PR:PROJECT_SUMMARY               	the ferroptosis-resistant phenotype via directly targeting SOX13 and promoting
PR:PROJECT_SUMMARY               	TRIM25-mediated ubiquitination and degradation of SOX13. Here we show,
PR:PROJECT_SUMMARY               	SOX13/SCAF1 are important in ferroptosis-resistance, and targeting SOX13 with
PR:PROJECT_SUMMARY               	zanamivir has therapeutic potential. We conducted untargeted metabolomic
PR:PROJECT_SUMMARY               	analysis of Erastin-resis SNU-668 cells transfected with shRNA-SOX13 or
PR:PROJECT_SUMMARY               	shRNA-NC.
PR:INSTITUTE                     	Fudan university shanghai cancer center
PR:DEPARTMENT                    	Department of Gastric Surgery
PR:LAST_NAME                     	Mingzhe
PR:FIRST_NAME                    	Ma
PR:ADDRESS                       	building 18, 29 nong linling road, xuhui district, shanghai, 200024, China
PR:EMAIL                         	mmz666@163.com, ding@bioinformatics.com.cn
PR:PHONE                         	13917006049
#STUDY
ST:STUDY_TITLE                   	Targeting SOX13 inhibits the assembly of respiratory chain supercomplexes to
ST:STUDY_TITLE                   	overcome ferroptosis-resistance in gastric cancer
ST:STUDY_SUMMARY                 	Therapeutic resistance represents a bottleneck to treatment in advanced gastric
ST:STUDY_SUMMARY                 	cancer (GC). Ferroptosis is an iron-dependent form of non-apoptotic cell death
ST:STUDY_SUMMARY                 	and is associated with anti-cancer therapeutic efficacy. Further investigations
ST:STUDY_SUMMARY                 	are required to clarify the underlying mechanisms. Ferroptosis-resistant GC cell
ST:STUDY_SUMMARY                 	lines are constructed. Dysregulated mRNAs between ferroptosis-resistant and
ST:STUDY_SUMMARY                 	parental cell lines are identified. The expression of SOX13/SCAF1 is manipulated
ST:STUDY_SUMMARY                 	in GC cell lines where relevant biological and molecular analyses are performed.
ST:STUDY_SUMMARY                 	Molecular docking and computational screening are performed to screen potential
ST:STUDY_SUMMARY                 	inhibitors of SOX13. We show that SOX13 boosts protein remodeling of electron
ST:STUDY_SUMMARY                 	transport chain (ETC) complexes by directly transactivating SCAF1. This leads to
ST:STUDY_SUMMARY                 	increased supercomplexes (SCs) assembly, mitochondrial respiration,
ST:STUDY_SUMMARY                 	mitochondrial energetics and chemo- and immune-resistance. Zanamivir, reverts
ST:STUDY_SUMMARY                 	the ferroptosis-resistant phenotype via directly targeting SOX13 and promoting
ST:STUDY_SUMMARY                 	TRIM25-mediated ubiquitination and degradation of SOX13. Here we show,
ST:STUDY_SUMMARY                 	SOX13/SCAF1 are important in ferroptosis-resistance, and targeting SOX13 with
ST:STUDY_SUMMARY                 	zanamivir has therapeutic potential. We conducted untargeted metabolomic
ST:STUDY_SUMMARY                 	analysis of Erastin-resis SNU-668 cells transfected with shRNA-SOX13 or
ST:STUDY_SUMMARY                 	shRNA-NC.
ST:INSTITUTE                     	Fudan university shanghai cancer center
ST:LAST_NAME                     	Ma
ST:FIRST_NAME                    	Mingzhe
ST:ADDRESS                       	lingling road, xuhui district, shanghai, China
ST:EMAIL                         	mmz666@163.com, ding@bioinformatics.com.cn
ST:PHONE                         	13917006049
#SUBJECT
SU:SUBJECT_TYPE                  	Human
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           	-	NC-1	Sample source:Erastin-resis SNU-668 cells | transfected:shRNA-NC	RAW_FILE_NAME(Raw file name)=NC-1.raw
SUBJECT_SAMPLE_FACTORS           	-	NC-2	Sample source:Erastin-resis SNU-669 cells | transfected:shRNA-NC	RAW_FILE_NAME(Raw file name)=NC-2.raw
SUBJECT_SAMPLE_FACTORS           	-	NC-3	Sample source:Erastin-resis SNU-670 cells | transfected:shRNA-NC	RAW_FILE_NAME(Raw file name)=NC-3.raw
SUBJECT_SAMPLE_FACTORS           	-	NC-4	Sample source:Erastin-resis SNU-671 cells | transfected:shRNA-NC	RAW_FILE_NAME(Raw file name)=NC-4.raw
SUBJECT_SAMPLE_FACTORS           	-	NC-5	Sample source:Erastin-resis SNU-672 cells | transfected:shRNA-NC	RAW_FILE_NAME(Raw file name)=NC-5.raw
SUBJECT_SAMPLE_FACTORS           	-	NC-6	Sample source:Erastin-resis SNU-673 cells | transfected:shRNA-NC	RAW_FILE_NAME(Raw file name)=NC-6.raw
SUBJECT_SAMPLE_FACTORS           	-	NC-7	Sample source:Erastin-resis SNU-674 cells | transfected:shRNA-NC	RAW_FILE_NAME(Raw file name)=NC-7.raw
SUBJECT_SAMPLE_FACTORS           	-	NC-8	Sample source:Erastin-resis SNU-675 cells | transfected:shRNA-NC	RAW_FILE_NAME(Raw file name)=NC-8.raw
SUBJECT_SAMPLE_FACTORS           	-	NC-9	Sample source:Erastin-resis SNU-676 cells | transfected:shRNA-NC	RAW_FILE_NAME(Raw file name)=NC-9.raw
SUBJECT_SAMPLE_FACTORS           	-	NC-10	Sample source:Erastin-resis SNU-677 cells | transfected:shRNA-NC	RAW_FILE_NAME(Raw file name)=NC-10.raw
SUBJECT_SAMPLE_FACTORS           	-	NC-11	Sample source:Erastin-resis SNU-678 cells | transfected:shRNA-NC	RAW_FILE_NAME(Raw file name)=NC-11.raw
SUBJECT_SAMPLE_FACTORS           	-	NC-12	Sample source:Erastin-resis SNU-679 cells | transfected:shRNA-NC	RAW_FILE_NAME(Raw file name)=NC-12.raw
SUBJECT_SAMPLE_FACTORS           	-	Sox13-1	Sample source:Erastin-resis SNU-680 cells | transfected:shRNA-SOX13	RAW_FILE_NAME(Raw file name)=Sox13-1.raw
SUBJECT_SAMPLE_FACTORS           	-	Sox13-2	Sample source:Erastin-resis SNU-681 cells | transfected:shRNA-SOX14	RAW_FILE_NAME(Raw file name)=Sox13-2.raw
SUBJECT_SAMPLE_FACTORS           	-	Sox13-3	Sample source:Erastin-resis SNU-682 cells | transfected:shRNA-SOX15	RAW_FILE_NAME(Raw file name)=Sox13-3.raw
SUBJECT_SAMPLE_FACTORS           	-	Sox13-4	Sample source:Erastin-resis SNU-683 cells | transfected:shRNA-SOX16	RAW_FILE_NAME(Raw file name)=Sox13-4.raw
SUBJECT_SAMPLE_FACTORS           	-	Sox13-5	Sample source:Erastin-resis SNU-684 cells | transfected:shRNA-SOX17	RAW_FILE_NAME(Raw file name)=Sox13-5.raw
SUBJECT_SAMPLE_FACTORS           	-	Sox13-6	Sample source:Erastin-resis SNU-685 cells | transfected:shRNA-SOX18	RAW_FILE_NAME(Raw file name)=Sox13-6.raw
SUBJECT_SAMPLE_FACTORS           	-	Sox13-7	Sample source:Erastin-resis SNU-686 cells | transfected:shRNA-SOX19	RAW_FILE_NAME(Raw file name)=Sox13-7.raw
SUBJECT_SAMPLE_FACTORS           	-	Sox13-8	Sample source:Erastin-resis SNU-687 cells | transfected:shRNA-SOX20	RAW_FILE_NAME(Raw file name)=Sox13-8.raw
SUBJECT_SAMPLE_FACTORS           	-	Sox13-9	Sample source:Erastin-resis SNU-688 cells | transfected:shRNA-SOX21	RAW_FILE_NAME(Raw file name)=Sox13-9.raw
SUBJECT_SAMPLE_FACTORS           	-	Sox13-10	Sample source:Erastin-resis SNU-689 cells | transfected:shRNA-SOX22	RAW_FILE_NAME(Raw file name)=Sox13-10.raw
SUBJECT_SAMPLE_FACTORS           	-	Sox13-11	Sample source:Erastin-resis SNU-668 cells | transfected:shRNA-SOX23	RAW_FILE_NAME(Raw file name)=Sox13-11.raw
SUBJECT_SAMPLE_FACTORS           	-	Sox13-12	Sample source:Erastin-resis SNU-669 cells | transfected:shRNA-SOX24	RAW_FILE_NAME(Raw file name)=Sox13-12.raw
#COLLECTION
CO:COLLECTION_SUMMARY            	SNU-668 Erastin-resistant cells were were cultured for 48-72 h in advanced
CO:COLLECTION_SUMMARY            	RPMI-1640 medium (Gibco) without supplements.
CO:SAMPLE_TYPE                   	SNU-668 Erastin-resistant cells
#TREATMENT
TR:TREATMENT_SUMMARY             	Erastin-resis SNU-668 cells transfected with shRNA-NC or shRNA-SOX13
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	For untargeted metabolomics, a total of 24 samples were analyzed (n=12
SP:SAMPLEPREP_SUMMARY            	Erastinresis SNU-668 cells transfected with shRNA-NC, n=12 Erastinresis SNU-668
SP:SAMPLEPREP_SUMMARY            	cells transfected with shRNA-SOX13). 2 × 105 cells of adherent cells were
SP:SAMPLEPREP_SUMMARY            	harvested in six-well plates. When collected, cells were washed by cold PBS
SP:SAMPLEPREP_SUMMARY            	buffer twice and immediately quenched in liquid nitrogen. Tumor samples were
SP:SAMPLEPREP_SUMMARY            	weighed and pulverized. All samples were lysed in 1 ml of −80°C extraction
SP:SAMPLEPREP_SUMMARY            	solvent (80% methanol/water). After centrifugation (20,000g, 4°C, 15 min),
SP:SAMPLEPREP_SUMMARY            	supernatant was transferred to a new tube, and samples were dried using a vacuum
SP:SAMPLEPREP_SUMMARY            	centrifugal concentrator. Blood samples from patients and mice were collected
SP:SAMPLEPREP_SUMMARY            	into BD Vacutainer blood collection tubes and placed on ice. Serum was isolated
SP:SAMPLEPREP_SUMMARY            	by centrifugation (15,000g, 4°C, 10 min), and aliquots of 100 μl of
SP:SAMPLEPREP_SUMMARY            	supernatant were frozen immediately at −80°C. Metabolites were reconstituted
SP:SAMPLEPREP_SUMMARY            	in 150 μl of 80% acetonitrile/water, vortexed, and centrifuged to remove
SP:SAMPLEPREP_SUMMARY            	insoluble material. All samples were stored at −80°C before LC-MS/MS
SP:SAMPLEPREP_SUMMARY            	analysis.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Samples were separated on an amide column, using mobile phase A consists of
CH:CHROMATOGRAPHY_SUMMARY        	water mixed with 25 mM ammonium acetate and 25 mM Ammonium hydroxide and mobile
CH:CHROMATOGRAPHY_SUMMARY        	phase B ACN. The injection volume was 4 µL and flow rate was 0.4 ml/min. 1. The
CH:CHROMATOGRAPHY_SUMMARY        	generic HPLC gradient was listed in Table 1: 2.
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Agilent 1260
CH:COLUMN_NAME                   	Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um)
CH:SOLVENT_A                     	100% water; 25mM ammonium acetate; 25mM ammonium hydroxide
CH:SOLVENT_B                     	100% acetonitrile
CH:FLOW_GRADIENT                 	0.0 min 10% 1.0 min 10% 11.0 min 13% 14.0 min 20% 16.5 min 30% 18.5 min 50% 20.5
CH:FLOW_GRADIENT                 	min 80% 25.0 min 80% 25.1 min 10% 34.0 min 10%
CH:FLOW_RATE                     	0.4 ml/min
CH:COLUMN_TEMPERATURE            	350
#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                   	S analysis was carried out on the Q-Exactive MS/MS in both positive and negative
MS:MS_COMMENTS                   	ion modes. 1) Set the relevant tuning parameters for the probe as listed: aux
MS:MS_COMMENTS                   	gas heater temperature, 400 °C; sheath gas, 40; auxiliary gas, 13; spray
MS:MS_COMMENTS                   	voltage, 3.5 kV for positive mode and negative mode. Set the capillary
MS:MS_COMMENTS                   	temperature at 350 °C, and S-lens at 55. 2) Build a DDA method as follows: Full
MS:MS_COMMENTS                   	scan range: 60 to 900 (m/z); resolution for MS1 and ddMS2: 70,000 and 17,500
MS:MS_COMMENTS                   	respectively; maximum injection time for MS1 and ddMS2: 100 ms and 45 ms;
MS:MS_COMMENTS                   	automatic gain control (AGC) for MS1 and ddMS2: 3e6 and 2e5; isolation window:
MS:MS_COMMENTS                   	1.6 m/z; normalized collision energies (NCE): 10, 17, 25 or 30, 40, 50. 3) Build
MS:MS_COMMENTS                   	a full scan method as follows: Full scan range: 60 to 900 (m/z); resolution:
MS:MS_COMMENTS                   	140,000; maximum injection time: 100ms; automatic gain control (AGC): 3e6 ions.
MS:MS_COMMENTS                   	Raw files were submitted to Thermo Compound Discover 2.1, (CD), and processed
MS:MS_COMMENTS                   	with Untargeted Metabolomics workflow with minor modification to find and
MS:MS_COMMENTS                   	identify the differences between samples: Performs retention time alignment,
MS:MS_COMMENTS                   	unknown compound detection, and compound grouping across all samples. Predicts
MS:MS_COMMENTS                   	elemental compositions for all compounds, fills gaps across all sam ples, and
MS:MS_COMMENTS                   	hides chemical background (using Blank samples). Identifies compounds using
MS:MS_COMMENTS                   	mzCloud (ddMS2) and ChemSpider (formula or exact mass). Also performs similarity
MS:MS_COMMENTS                   	search for all com pounds with ddMS2 data using mzCloud. Maps compounds to
MS:MS_COMMENTS                   	biological pathways using KEGG database For retention time alignment, the max
MS:MS_COMMENTS                   	time shift was 2 mins, and a tolerance of 0.5 min was used for grouping unknown
MS:MS_COMMENTS                   	compounds. Mass tolerance were set as 10 ppm for feature detection and 5 ppm for
MS:MS_COMMENTS                   	compound annotation. The exact mass of each feature was submitted to ChemSpider
MS:MS_COMMENTS                   	with 4 databases selected (BioCyc; Human Metabolome Database; KEGG; LipidMAPS).
MS:MS_COMMENTS                   	Results from Compound Discover, the compound table, was exported as .xsls file,
MS:MS_COMMENTS                   	and then analysed with R.
MS:MS_RESULTS_FILE               	ST003134_AN005144_Results.txt	UNITS:m/z	Has m/z:Yes	Has RT:No	RT units:No RT data
#END