#METABOLOMICS WORKBENCH weishengxin_20250529_221928 DATATRACK_ID:5972 STUDY_ID:ST003953 ANALYSIS_ID:AN006498 PROJECT_ID:PR002476
VERSION             	1
CREATED_ON             	June 7, 2025, 6:15 am
#PROJECT
PR:PROJECT_TITLE                 	Targeting ESRP1 inhibits the assembly of respiratory chain supercomplexes to
PR:PROJECT_TITLE                 	overcome ferroptosis-resistance in gastric cancer
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 ESRP1/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 ESRP1. We show that ESRP1 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 ESRP1 and promoting
PR:PROJECT_SUMMARY               	TRIM25-mediated ubiquitination and degradation of ESRP1. Here we show,
PR:PROJECT_SUMMARY               	ESRP1/SCAF1 are important in ferroptosis-resistance, and targeting ESRP1 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-ESRP1 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                   	Metabolomic Profiling of Gastric Cancer Post-ESRP1 Gene Knockout
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 ESRP1/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 ESRP1. We show that ESRP1 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 ESRP1 and promoting
ST:STUDY_SUMMARY                 	TRIM25-mediated ubiquitination and degradation of ESRP1. Here we show,
ST:STUDY_SUMMARY                 	ESRP1/SCAF1 are important in ferroptosis-resistance, and targeting ESRP1 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-ESRP1 or
ST:STUDY_SUMMARY                 	shRNA-NC.
ST:INSTITUTE                     	Fudan university shanghai cancer center
ST:DEPARTMENT                    	Department of Gastric Surgery
ST:LAST_NAME                     	Mingzhe
ST:FIRST_NAME                    	Ma
ST:ADDRESS                       	building 18, 29 Nong Linling Road, Xuhui district, Shanghai, 200024, China
ST:EMAIL                         	mmz666@163.com, ding@bioinformatics.com.cn
ST:PHONE                         	13917006049
ST:NUM_GROUPS                    	2
ST:TOTAL_SUBJECTS                	2
#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           	-	siESRP1_1	Sample source:siESRP1_1 | Genotype:Knockout | Treatment:Knockout	RAW_FILE_NAME(Raw file name NEG)=NEG_Gg25JS_ESRP1_1.raw; RAW_FILE_NAME(Raw file name POS)=POS_Gg25JS_ESRP1_1.raw
SUBJECT_SAMPLE_FACTORS           	-	siESRP1_2	Sample source:siESRP1_2 | Genotype:Knockout | Treatment:Knockout	RAW_FILE_NAME(Raw file name NEG)=NEG_Gg25JS_ESRP1_2.raw; RAW_FILE_NAME(Raw file name POS)=POS_Gg25JS_ESRP1_2.raw
SUBJECT_SAMPLE_FACTORS           	-	siESRP1_3	Sample source:siESRP1_3 | Genotype:Knockout | Treatment:Knockout	RAW_FILE_NAME(Raw file name NEG)=NEG_Gg25JS_ESRP1_3.raw; RAW_FILE_NAME(Raw file name POS)=POS_Gg25JS_ESRP1_3.raw
SUBJECT_SAMPLE_FACTORS           	-	siESRP1_4	Sample source:siESRP1_4 | Genotype:Knockout | Treatment:Knockout	RAW_FILE_NAME(Raw file name NEG)=NEG_Gg25JS_ESRP1_4.raw; RAW_FILE_NAME(Raw file name POS)=POS_Gg25JS_ESRP1_4.raw
SUBJECT_SAMPLE_FACTORS           	-	siESRP1_5	Sample source:siESRP1_5 | Genotype:Knockout | Treatment:Knockout	RAW_FILE_NAME(Raw file name NEG)=NEG_Gg25JS_ESRP1_5.raw; RAW_FILE_NAME(Raw file name POS)=POS_Gg25JS_ESRP1_5.raw
SUBJECT_SAMPLE_FACTORS           	-	siESRP1_6	Sample source:siESRP1_6 | Genotype:Knockout | Treatment:Knockout	RAW_FILE_NAME(Raw file name NEG)=NEG_Gg25JS_ESRP1_6.raw; RAW_FILE_NAME(Raw file name POS)=POS_Gg25JS_ESRP1_6.raw
SUBJECT_SAMPLE_FACTORS           	-	NC1	Sample source:NC1 | Genotype:Control | Treatment:Control	RAW_FILE_NAME(Raw file name NEG)=NEG_Gg25JS_NC1.raw; RAW_FILE_NAME(Raw file name POS)=POS_Gg25JS_NC1.raw
SUBJECT_SAMPLE_FACTORS           	-	NC2	Sample source:NC2 | Genotype:Control | Treatment:Control	RAW_FILE_NAME(Raw file name NEG)=NEG_Gg25JS_NC2.raw; RAW_FILE_NAME(Raw file name POS)=POS_Gg25JS_NC2.raw
SUBJECT_SAMPLE_FACTORS           	-	NC3	Sample source:NC3 | Genotype:Control | Treatment:Control	RAW_FILE_NAME(Raw file name NEG)=NEG_Gg25JS_NC3.raw; RAW_FILE_NAME(Raw file name POS)=POS_Gg25JS_NC3.raw
SUBJECT_SAMPLE_FACTORS           	-	NC4	Sample source:NC4 | Genotype:Control | Treatment:Control	RAW_FILE_NAME(Raw file name NEG)=NEG_Gg25JS_NC4.raw; RAW_FILE_NAME(Raw file name POS)=POS_Gg25JS_NC4.raw
SUBJECT_SAMPLE_FACTORS           	-	NC5	Sample source:NC5 | Genotype:Control | Treatment:Control	RAW_FILE_NAME(Raw file name NEG)=NEG_Gg25JS_NC5.raw; RAW_FILE_NAME(Raw file name POS)=POS_Gg25JS_NC5.raw
SUBJECT_SAMPLE_FACTORS           	-	NC6	Sample source:NC6 | Genotype:Control | Treatment:Control	RAW_FILE_NAME(Raw file name NEG)=NEG_Gg25JS_NC6.raw; RAW_FILE_NAME(Raw file name POS)=POS_Gg25JS_NC6.raw
#COLLECTION
CO:COLLECTION_SUMMARY            	Erastinresis SNU-668 cells transfected with shRNA-NC, n = 12 Erastin-resis
CO:COLLECTION_SUMMARY            	SNU-668 cells transfected with shRNA-ESRP1 or shRNA-NC. 2 × 105 cells of
CO:COLLECTION_SUMMARY            	adherent cells were harvested in six-well plates. When collected,
CO:COLLECTION_SUMMARY            	cellswerewashed by cold PBS buffer twice and immediately quenched in liquid
CO:COLLECTION_SUMMARY            	nitrogen. Tumor samples were weighed and pulverized. All samples were lysed in
CO:COLLECTION_SUMMARY            	1ml of −80 °C extraction solvent (80% methanol/ water). After centrifugation
CO:COLLECTION_SUMMARY            	(20,000 g, 4 °C, 15min), supernatant was transferred to a new tube, and samples
CO:COLLECTION_SUMMARY            	were dried using a vacuum centrifugal concentrator. Blood samples from patients
CO:COLLECTION_SUMMARY            	and mice were collected into BD Vacutainer blood collection tubes and placed on
CO:COLLECTION_SUMMARY            	ice. Serum was isolated by centrifugation (15,000 g, 4 °C, 10min), and aliquots
CO:COLLECTION_SUMMARY            	of 100 μl of supernatant were frozen immediately at −80 °C. Metabolites were
CO:COLLECTION_SUMMARY            	reconstituted in 150 μl of 80% acetonitrile/water, vortexed, and centrifuged to
CO:COLLECTION_SUMMARY            	remove insoluble material. All samples were stored at −80 °C before LC-MS/MS
CO:COLLECTION_SUMMARY            	analysis.
CO:SAMPLE_TYPE                   	SNU-668 Erastin-resistant cells
CO:STORAGE_CONDITIONS            	-80℃
#TREATMENT
TR:TREATMENT_SUMMARY             	Stable knockdown of ESRP1 in GC cell lines was generated by lentiviral-based
TR:TREATMENT_SUMMARY             	shRNA delivery. Specific target shRNAs were subcloned into lentiviral vector
TR:TREATMENT_SUMMARY             	pLKO.1 (Shanghai Genechem), while a non-target shRNA was used as a negative
TR:TREATMENT_SUMMARY             	control. The shRNAs were designed and synthesized by GenePharma (Shanghai,
TR:TREATMENT_SUMMARY             	China) to suppress the gene expression. The design of the shRNAs was assisted by
TR:TREATMENT_SUMMARY             	the use of web-based software provided by Invitrogen
TR:TREATMENT_SUMMARY             	(http://rnaidesigner.invitrogen.com/rnaiexpress/). Blast searches were performed
TR:TREATMENT_SUMMARY             	using the National Center for Biotechnology Information expressed sequence tag
TR:TREATMENT_SUMMARY             	database to ensure that the shRNA construct only targeted human ESRP1
TR:TREATMENT_SUMMARY             	expression. Erastin-resis SNU-668 cells transfected with shRNA-NC, or
TR:TREATMENT_SUMMARY             	shRNA-ESRP1.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	For untargeted metabolomics, a total of 12 samples were analyzed (n=6
SP:SAMPLEPREP_SUMMARY            	Erastinresis SNU-668 cells transfected with shRNA-NC, n=12 Erastinresis SNU-668
SP:SAMPLEPREP_SUMMARY            	cells transfected with shRNA-ESRP1). Transfer all samples to a 2 mL centrifuge
SP:SAMPLEPREP_SUMMARY            	tube and add one grinding bead with a diameter of 6 mm. Add 200 µL of
SP:SAMPLEPREP_SUMMARY            	extraction solution (methanol:water = 4:1 (v:v)), containing four internal
SP:SAMPLEPREP_SUMMARY            	standards (e.g., L-2-chlorophenylalanine at 0.02 mg/mL). Grind using a cryogenic
SP:SAMPLEPREP_SUMMARY            	tissue grinder for 6 minutes (-10°C, 50 Hz). Perform low-temperature ultrasonic
SP:SAMPLEPREP_SUMMARY            	extraction for 30 minutes (5°C, 40 kHz). Incubate the samples at -20°C for 30
SP:SAMPLEPREP_SUMMARY            	minutes. Centrifuge for 15 minutes (13,000 g, 4°C), and transfer the
SP:SAMPLEPREP_SUMMARY            	supernatant to an injection vial with an insert for analysis. Additionally,
SP:SAMPLEPREP_SUMMARY            	transfer 20 µL of the supernatant from each sample into a separate container to
SP:SAMPLEPREP_SUMMARY            	create a pooled quality control sample.
SP:PROCESSING_STORAGE_CONDITIONS 	-80℃
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Waters Acquity H-Class
CH:COLUMN_NAME                   	Waters ACQUITY UPLC HSS T3 (100 x 2.1 mm, 1.8 µm)
CH:SOLVENT_A                     	95% Water/5% Acetonitrile; 0.1% Formic acid
CH:SOLVENT_B                     	47.5% Acetonitrile/47.5% Isopropanol/5% Water; 0.1% Formic acid
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_RATE                     	0.4 mL/min
CH:COLUMN_TEMPERATURE            	40°C
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive HF-X Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	The pretreatment of LC/MS raw data was performed by Progenesis QI (Waters
MS:MS_COMMENTS                   	Corporation,Milford,USA) software, and a three-dimensional data matrixin CSV
MS:MS_COMMENTS                   	format was exported.The information in this three-dimensional matrix
MS:MS_COMMENTS                   	included:sample information,metabolite name and mass spectral response
MS:MS_COMMENTS                   	intensity. Internal standard peaks,as well as any known false positive peaks
MS:MS_COMMENTS                   	(includingnoise,column bleed,and derivatized reagent peaks), were removed from
MS:MS_COMMENTS                   	the datamatrix,deredundant and peak pooled. At the same time, the metabolites
MS:MS_COMMENTS                   	wereidentified by searching database,and the main databases were the
MS:MS_COMMENTS                   	HMDB(http://www.hmdb.ca/),Metlin(https:/metlin.scripps.edu/)and the
MS:MS_COMMENTS                   	self-compiledMajorbioDatabase(MJDB)of Majorbio Biotechnology
MS:MS_COMMENTS                   	Co.,Ltd.(Shanghai,China)
MS:MS_RESULTS_FILE               	ST003953_AN006498_Results.txt	UNITS:peak intensity	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END