#METABOLOMICS WORKBENCH kconge2_20240617_174608 DATATRACK_ID:4936 STUDY_ID:ST003306 ANALYSIS_ID:AN005417 PROJECT_ID:PR002056
VERSION             	1
CREATED_ON             	July 9, 2024, 12:26 pm
#PROJECT
PR:PROJECT_TITLE                 	ASCT2 is a major contributor to serine uptake in cancer cells
PR:PROJECT_TYPE                  	GCMS quantitative analysis
PR:PROJECT_SUMMARY               	Polar metabolite abundance studies from MCF7 human ER+ breast cancer cell line
PR:PROJECT_SUMMARY               	with or without CRISPR-Cas9 knockout of ASCT2. Studies used RPMI media with
PR:PROJECT_SUMMARY               	either complete serine levels (285uM), low serine levels (50uM), or no
PR:PROJECT_SUMMARY               	glutamine.
PR:INSTITUTE                     	University of Illinois Chicago
PR:DEPARTMENT                    	Physiology and Biophysics
PR:LABORATORY                    	Coloff Lab
PR:LAST_NAME                     	Conger
PR:FIRST_NAME                    	Kelly
PR:ADDRESS                       	909 S Wolcott Ave, Chicago, IL, 60612
PR:EMAIL                         	kconge2@uic.edu
PR:PHONE                         	2314320406
#STUDY
ST:STUDY_TITLE                   	ASCT2 is a major contributor to serine uptake in cancer cells
ST:STUDY_SUMMARY                 	The non-essential amino acid serine is a critical nutrient for cancer cells due
ST:STUDY_SUMMARY                 	to its diverse biosynthetic functions. While some tumors can synthesize serine
ST:STUDY_SUMMARY                 	de novo, others are auxotrophic and therefore reliant on serine uptake.
ST:STUDY_SUMMARY                 	Importantly, despite several transporters being known to be capable of
ST:STUDY_SUMMARY                 	transporting serine, the transporter(s) that mediate serine uptake in cancer
ST:STUDY_SUMMARY                 	cells are not known. Here, we characterize the amino acid transporter ASCT2
ST:STUDY_SUMMARY                 	(SLC1A5) as a major contributor to serine uptake in cancer cells. ASCT2 is
ST:STUDY_SUMMARY                 	well-known as a glutamine transporter in cancer, and our work demonstrates that
ST:STUDY_SUMMARY                 	serine and glutamine compete for uptake through ASCT2. We further show that
ST:STUDY_SUMMARY                 	ASCT2-mediated serine uptake is essential for purine nucleotide biosynthesis and
ST:STUDY_SUMMARY                 	that ERα promotes serine uptake by directly activating SLC1A5 transcription.
ST:STUDY_SUMMARY                 	Together, our work defines an additional important role for ASCT2 as a serine
ST:STUDY_SUMMARY                 	transporter in cancer and evaluates ASCT2 as a potential therapeutic target.
ST:INSTITUTE                     	University of Illinois Chicago
ST:DEPARTMENT                    	Physiology and Biophysics
ST:LABORATORY                    	Coloff Lab
ST:LAST_NAME                     	Conger
ST:FIRST_NAME                    	Kelly
ST:ADDRESS                       	909 S Wolcott Ave, Chicago, IL, 60612
ST:EMAIL                         	kconge2@uic.edu
ST:PHONE                         	2314320406
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
SU:GENDER                        	Female
SU:CELL_STRAIN_DETAILS           	MCF7
SU:CELL_PRIMARY_IMMORTALIZED     	Immortalized
#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           	sgLuc IM 1	sgLuc IM 1	Sample source:Human breast cancer cells | Genotype:Ctrl | Treatment:50uM Serine	Cell line=MCF7; RAW_FILE_NAME(Raw_File_Name)=sgLuc IM 1.D
SUBJECT_SAMPLE_FACTORS           	sgLuc IM 2	sgLuc IM 2	Sample source:Human breast cancer cells | Genotype:Ctrl | Treatment:50uM Serine	Cell line=MCF7; RAW_FILE_NAME(Raw_File_Name)=sgLuc IM 2.D
SUBJECT_SAMPLE_FACTORS           	sgLuc IM 3	sgLuc IM 3	Sample source:Human breast cancer cells | Genotype:Ctrl | Treatment:50uM Serine	Cell line=MCF7; RAW_FILE_NAME(Raw_File_Name)=sgLuc IM 3.D
SUBJECT_SAMPLE_FACTORS           	sgSLC1A5-1 IM 1	sgSLC1A5-1 IM 1	Sample source:Human breast cancer cells | Genotype:ASCT2-KO | Treatment:50uM Serine	Cell line=MCF7; RAW_FILE_NAME(Raw_File_Name)=sgSLC1A5-1 IM 1.D
SUBJECT_SAMPLE_FACTORS           	sgSLC1A5-1 IM 2	sgSLC1A5-1 IM 2	Sample source:Human breast cancer cells | Genotype:ASCT2-KO | Treatment:50uM Serine	Cell line=MCF7; RAW_FILE_NAME(Raw_File_Name)=sgSLC1A5-1 IM 2.D
SUBJECT_SAMPLE_FACTORS           	sgSLC1A5-1 IM 3	sgSLC1A5-1 IM 3	Sample source:Human breast cancer cells | Genotype:ASCT2-KO | Treatment:50uM Serine	Cell line=MCF7; RAW_FILE_NAME(Raw_File_Name)=sgSLC1A5-1 IM 3.D
SUBJECT_SAMPLE_FACTORS           	sgSLC1A5-3 IM 1	sgSLC1A5-3 IM 1	Sample source:Human breast cancer cells | Genotype:ASCT2-KO | Treatment:50uM Serine	Cell line=MCF7; RAW_FILE_NAME(Raw_File_Name)=sgSLC1A5-3 IM 1.D
SUBJECT_SAMPLE_FACTORS           	sgSLC1A5-3 IM 2	sgSLC1A5-3 IM 2	Sample source:Human breast cancer cells | Genotype:ASCT2-KO | Treatment:50uM Serine	Cell line=MCF7; RAW_FILE_NAME(Raw_File_Name)=sgSLC1A5-3 IM 2.D
SUBJECT_SAMPLE_FACTORS           	sgSLC1A5-3 IM 3	sgSLC1A5-3 IM 3	Sample source:Human breast cancer cells | Genotype:ASCT2-KO | Treatment:50uM Serine	Cell line=MCF7; RAW_FILE_NAME(Raw_File_Name)=sgSLC1A5-3 IM 3.D
SUBJECT_SAMPLE_FACTORS           	Luc Puro 1	Luc Puro 1	Sample source:Human breast cancer cells | Genotype:Ctrl | Treatment:285uM Serine	Cell line=MCF7; RAW_FILE_NAME(Raw_File_Name)=sgLuc Puro 1.D
SUBJECT_SAMPLE_FACTORS           	Luc Puro 2	Luc Puro 2	Sample source:Human breast cancer cells | Genotype:Ctrl | Treatment:285uM Serine	Cell line=MCF7; RAW_FILE_NAME(Raw_File_Name)=sgLuc Puro 2.D
SUBJECT_SAMPLE_FACTORS           	Luc Puro 3	Luc Puro 3	Sample source:Human breast cancer cells | Genotype:Ctrl | Treatment:285uM Serine	Cell line=MCF7; RAW_FILE_NAME(Raw_File_Name)=sgLuc Puro 3.D
SUBJECT_SAMPLE_FACTORS           	SLC1A5-1,1	SLC1A5-1,1	Sample source:Human breast cancer cells | Genotype:ASCT2-KO | Treatment:285uM Serine	Cell line=MCF7; RAW_FILE_NAME(Raw_File_Name)=sgSLC1A5-1,1.D
SUBJECT_SAMPLE_FACTORS           	SLC1A5-1,2	SLC1A5-1,2	Sample source:Human breast cancer cells | Genotype:ASCT2-KO | Treatment:285uM Serine	Cell line=MCF7; RAW_FILE_NAME(Raw_File_Name)=sgSLC1A5-1,2.D
SUBJECT_SAMPLE_FACTORS           	SLC1A5-1,3	SLC1A5-1,3	Sample source:Human breast cancer cells | Genotype:ASCT2-KO | Treatment:285uM Serine	Cell line=MCF7; RAW_FILE_NAME(Raw_File_Name)=sgSLC1A5-1,3.D
SUBJECT_SAMPLE_FACTORS           	SLC1A5-3,1	SLC1A5-3,1	Sample source:Human breast cancer cells | Genotype:ASCT2-KO | Treatment:285uM Serine	Cell line=MCF7; RAW_FILE_NAME(Raw_File_Name)=sgSLC1A5-3,1.D
SUBJECT_SAMPLE_FACTORS           	SLC1A5-3,2	SLC1A5-3,2	Sample source:Human breast cancer cells | Genotype:ASCT2-KO | Treatment:285uM Serine	Cell line=MCF7; RAW_FILE_NAME(Raw_File_Name)=sgSLC1A5-3,2.D
SUBJECT_SAMPLE_FACTORS           	SLC1A5-3,3	SLC1A5-3,3	Sample source:Human breast cancer cells | Genotype:ASCT2-KO | Treatment:285uM Serine	Cell line=MCF7; RAW_FILE_NAME(Raw_File_Name)=sgSLC1A5-3,3.D
SUBJECT_SAMPLE_FACTORS           	sgLuc -Q 60 1	sgLuc -Gln 60 1	Sample source:Human breast cancer cells | Genotype:Ctrl | Treatment:(-Gln)1hr	Cell line=MCF7; RAW_FILE_NAME(Raw_File_Name)=sgLuc -Q 60 1.D
SUBJECT_SAMPLE_FACTORS           	sgLuc -Q 60 2	sgLuc -Gln 60 2	Sample source:Human breast cancer cells | Genotype:Ctrl | Treatment:(-Gln)1hr	Cell line=MCF7; RAW_FILE_NAME(Raw_File_Name)=sgLuc -Q 60 2.D
SUBJECT_SAMPLE_FACTORS           	sgLuc -Q 60 3	sgLuc -Gln 60 3	Sample source:Human breast cancer cells | Genotype:Ctrl | Treatment:(-Gln)1hr	Cell line=MCF7; RAW_FILE_NAME(Raw_File_Name)=sgLuc -Q 60 3.D
#COLLECTION
CO:COLLECTION_SUMMARY            	Cells were washed with PBS and fed media containing either complete serine
CO:COLLECTION_SUMMARY            	(285uM) or low serine (50uM) for six hours. Cells were washed with saline and
CO:COLLECTION_SUMMARY            	harvested in cold GCMS-grade methanol with norvaline diluted in water. Polar
CO:COLLECTION_SUMMARY            	metabolites were extracted with the addition of chloroform. Samples were dried
CO:COLLECTION_SUMMARY            	under constant air flow for 2 hours.
CO:COLLECTION_PROTOCOL_FILENAME  	CL_PM2024.pdf
CO:SAMPLE_TYPE                   	Cultured cells
CO:STORAGE_CONDITIONS            	-80℃
#TREATMENT
TR:TREATMENT_SUMMARY             	Cells are either control (sgLuc) or ASCT2-KO (sgSLC1A5). All groups were plated
TR:TREATMENT_SUMMARY             	in triplicate in a six well plate 48hours prior to treatment. Cells were treated
TR:TREATMENT_SUMMARY             	with either complete media, or media containing low serine for six hours, or
TR:TREATMENT_SUMMARY             	media lacking glutamine (-Q) for one hour prior to harvesting.
TR:TREATMENT_DOSEDURATION        	1-6 hours
TR:CELL_GROWTH_CONTAINER         	Corning 6-well plates
TR:CELL_MEDIA                    	RPMI w/o Glucose, Sodium Pyruvate, Amino acids
TR:CELL_PCT_CONFLUENCE           	80%
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Samples were harvested in cold GCMS-grade methanol with norvaline diluted in
SP:SAMPLEPREP_SUMMARY            	water. Polar metabolites were separated with chloroform and air dried for two
SP:SAMPLEPREP_SUMMARY            	hours. Samples were then rehydrated in 15uL MOX reagent and heated at 37C for 90
SP:SAMPLEPREP_SUMMARY            	minutes. Then 20uL of TBDMS was added to each sample and heated at 60C for 60
SP:SAMPLEPREP_SUMMARY            	minutes.
SP:SAMPLEPREP_PROTOCOL_FILENAME  	CL_PM2024.pdf
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	All samples were analyzed by GC/MS using a HP-5MS Ultra Inert GC column
CH:CHROMATOGRAPHY_SUMMARY        	(19091S-433UI, Agilent Technologies) installed in an Agilent 7890B gas
CH:CHROMATOGRAPHY_SUMMARY        	chromatograph coupled to an Agilent 5779B mass spectrometer. Helium was used as
CH:CHROMATOGRAPHY_SUMMARY        	the carrier gas. One microliter was injected (split inlet) at 280 degrees C.
CH:CHROMATOGRAPHY_SUMMARY        	After injection, the GC oven was held at 60 degrees C for 1 minute before
CH:CHROMATOGRAPHY_SUMMARY        	ramping to 320 degrees C at 10C/min and held for 9 minutes at the maximum
CH:CHROMATOGRAPHY_SUMMARY        	temperature.
CH:CHROMATOGRAPHY_TYPE           	GC
CH:INSTRUMENT_NAME               	Agilent 7890B
CH:COLUMN_NAME                   	Agilent HP5-MS (30m x 0.25mm, 0.25 um)
CH:SOLVENT_A                     	N/A
CH:SOLVENT_B                     	N/A
CH:FLOW_GRADIENT                 	N/A
CH:FLOW_RATE                     	1.5mL/min
CH:COLUMN_TEMPERATURE            	60-320
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
AN:ANALYSIS_PROTOCOL_FILE        	CL_GCMS2024.pdf
#MS
MS:INSTRUMENT_NAME               	Agilent 5977B
MS:INSTRUMENT_TYPE               	Single quadrupole
MS:MS_TYPE                       	EI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	The MS system operated under electron impact ionization mode at 70 eV and the MS
MS:MS_COMMENTS                   	source and quadrupole were held at 230 degrees C and 150 degrees C respectively.
MS:MS_COMMENTS                   	Peak areas were determined using MassHunter software.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	Abundance
MS_METABOLITE_DATA_START
Samples	sgLuc IM 1	sgLuc IM 2	sgLuc IM 3	sgSLC1A5-1 IM 1	sgSLC1A5-1 IM 2	sgSLC1A5-1 IM 3	sgSLC1A5-3 IM 1	sgSLC1A5-3 IM 2	sgSLC1A5-3 IM 3
Factors	Sample source:Human breast cancer cells | Genotype:Ctrl | Treatment:50uM Serine	Sample source:Human breast cancer cells | Genotype:Ctrl | Treatment:50uM Serine	Sample source:Human breast cancer cells | Genotype:Ctrl | Treatment:50uM Serine	Sample source:Human breast cancer cells | Genotype:ASCT2-KO | Treatment:50uM Serine	Sample source:Human breast cancer cells | Genotype:ASCT2-KO | Treatment:50uM Serine	Sample source:Human breast cancer cells | Genotype:ASCT2-KO | Treatment:50uM Serine	Sample source:Human breast cancer cells | Genotype:ASCT2-KO | Treatment:50uM Serine	Sample source:Human breast cancer cells | Genotype:ASCT2-KO | Treatment:50uM Serine	Sample source:Human breast cancer cells | Genotype:ASCT2-KO | Treatment:50uM Serine
Norvaline	2697956	2303871	2267103	2524417	2202538	2289605	2484960	2364121	2281996
alpha-Ketoglutarate	10402	9888	9045	12673	9879	9472	11535	10254	10696
Aspartic Acid	423133	360546	344866	543128	476509	488161	609948	633544	559695
Citrate	36286	33583	32984	41730	34157	32315	44951	46076	42901
Citrulline	22268	20484	18261	15692	16168	17232	25030	15556	17513
Lactate	408296	409663	374741	341478	327207	292581	275397	275653	246223
L-Alanine	55694	51416	54068	129416	113097	112447	179333	187351	166781
L-Asparagine	346175	223916	205439	336078	272295	340272	484522	478085	374410
L-Cysteine	59575	38504	25806	27131	21751	24296	66706	50354	37462
L-Glutamate	1385417	1274150	1304122	1520223	1355443	1369860	1583715	1622297	1503952
L-Glutamine	1359422	1019394	952235	1149413	1107747	1085512	1501513	1523635	1164320
L-Glycine	298081	257184	263677	401319	339651	349507	413485	422620	387013
L-Isoleucine	713472	631024	577792	563223	495352	490266	688394	654675	596315
L-Leucine	792415	675603	640348	614344	550546	533982	758907	729609	648271
L-Lysine	21349	18126	17151	18113	15645	14640	23380	21443	18667
L-Methionine	80701	68333	63686	62289	54426	52746	73948	69724	63768
L-Phenylalanine	45170	37035	35797	34580	29476	28738	43748	40777	37147
L-Proline	63477	48227	49467	61301	59238	60450	73633	73136	64207
L-Serine	34549	28552	30886	22857	19633	20472	18978	15975	15181
L-Threonine	62029	48035	50976	40977	42288	51692	46303	51378	37526
L-Tyrosine	68229	61794	60444	55204	48238	48572	70472	66103	60378
L-Valine	270634	236044	222593	204315	183767	177932	266505	252465	224866
Malate	132722	120344	123263	143788	129266	128085	122554	126028	119490
OH-Proline	60748	55818	63224	63300	68379	76312	86080	99550	89039
Pyruvate	126663	112897	102740	111891	97328	93330	130419	117918	102525
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	PubChem ID
Norvaline	65098
alpha-Ketoglutarate	51
Aspartic Acid	5960
Citrate	31348
Citrulline	9750
Lactate	91435
L-Alanine	5950
L-Asparagine	6267
L-Cysteine	5862
L-Glutamate	33032
L-Glutamine	5961
L-Glycine	750
L-Isoleucine	6306
L-Leucine	6106
L-Lysine	5962
L-Methionine	6137
L-Phenylalanine	6140
L-Proline	145742
L-Serine	5951
L-Threonine	6288
L-Tyrosine	6057
L-Valine	6287
Malate	525
OH-Proline	614
Pyruvate	107735
METABOLITES_END
#END