#METABOLOMICS WORKBENCH juliehaines_20240918_073253 DATATRACK_ID:5206 STUDY_ID:ST003491 ANALYSIS_ID:AN005731 PROJECT_ID:PR002140
VERSION             	1
CREATED_ON             	September 18, 2024, 7:22 pm
#PROJECT
PR:PROJECT_TITLE                 	Blocking tryptophan catabolism reduces triple-negative breast cancer invasive
PR:PROJECT_TITLE                 	capacity
PR:PROJECT_SUMMARY               	Anchorage-independent triple-negative breast cancer (TNBC) cells exhibit
PR:PROJECT_SUMMARY               	elevated levels of the tryptophan (TRP) catabolizing enzyme tryptophan
PR:PROJECT_SUMMARY               	2,3-dioxygenase 2 (TDO2) compared to the same cells grown in two-dimensional
PR:PROJECT_SUMMARY               	culture. Tracing of 13C11-TRP demonstrated that anchorage-independent culture
PR:PROJECT_SUMMARY               	and/or inflammatory cytokines that activate nuclear factor
PR:PROJECT_SUMMARY               	kappa-light-chain-enhancer of activated B (NFκB) increase TRP catabolism and
PR:PROJECT_SUMMARY               	production of downstream catabolites such as kynurenine (KYN), which activate
PR:PROJECT_SUMMARY               	the aryl hydrocarbon receptor (AhR). TDO2 expression is heterogeneous within
PR:PROJECT_SUMMARY               	TNBC cell lines. To determine the function of TDO2, both pharmacologic
PR:PROJECT_SUMMARY               	inhibition and genetic manipulation were conducted. TDO2 knockdown revealed a
PR:PROJECT_SUMMARY               	compensatory increase in indoleamine 2,3-dioxygenase 1 (IDO1), a non-homologous
PR:PROJECT_SUMMARY               	TRP catabolizing enzyme, indicating that dual inhibition of these two enzymes is
PR:PROJECT_SUMMARY               	necessary to reliably block TRP catabolism. Thus, we tested a newly developed
PR:PROJECT_SUMMARY               	TDO2/IDO1 dual inhibitor, AT-0174, and found that it effectively inhibits TNBC
PR:PROJECT_SUMMARY               	TRP catabolism. Furthermore, AT-0174 treatment or AhR inhibitor significantly
PR:PROJECT_SUMMARY               	decreased TNBC anchorage-independent survival, invasive capacity, and expression
PR:PROJECT_SUMMARY               	of mesenchymal genes and protein, while exogenous KYN increased invasion through
PR:PROJECT_SUMMARY               	AhR-mediated ZEB1 expression. Thus, dual inhibition of TDO2/IDO1 may prove
PR:PROJECT_SUMMARY               	efficacious against TNBC progression.
PR:INSTITUTE                     	University of Colorado Anschutz Medical Campus
PR:LABORATORY                    	Lab of Angelo D'Alessandro in collaboration with lab of Jennifer Richer
PR:LAST_NAME                     	Haines
PR:FIRST_NAME                    	Julie
PR:ADDRESS                       	12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA
PR:EMAIL                         	julie.haines@cuanschutz.edu
PR:PHONE                         	3037243339
#STUDY
ST:STUDY_TITLE                   	Tryptophan/indole profiling of conditioned media from TNBC cell line SUM159PT
ST:STUDY_TITLE                   	with stable TDO2 overexpression.
ST:STUDY_SUMMARY                 	Conditioned media harvested from TNBC cell line SUM159PT +/- stable TDO2
ST:STUDY_SUMMARY                 	overexpression(OE) vs control (empty vector, EV).
ST:INSTITUTE                     	University of Colorado Anschutz Medical Campus
ST:LAST_NAME                     	Haines
ST:FIRST_NAME                    	Julie
ST:ADDRESS                       	12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA
ST:EMAIL                         	julie.haines@cuanschutz.edu
ST:PHONE                         	3037243339
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:GENDER                        	Female
#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           	-	SUM159-TDO2-1M	Sample source:Culture Media | factor:TDO2 overexpression	RAW_FILE_NAME(raw file name)=MR89-07
SUBJECT_SAMPLE_FACTORS           	-	SUM159-TDO2-2M	Sample source:Culture Media | factor:TDO2 overexpression	RAW_FILE_NAME(raw file name)=MR89-08
SUBJECT_SAMPLE_FACTORS           	-	SUM159-TDO2-3M	Sample source:Culture Media | factor:TDO2 overexpression	RAW_FILE_NAME(raw file name)=MR89-09
SUBJECT_SAMPLE_FACTORS           	-	SUM159-EV-1M	Sample source:Culture Media | factor:empty vector	RAW_FILE_NAME(raw file name)=MR89-10
SUBJECT_SAMPLE_FACTORS           	-	SUM159-EV-2M	Sample source:Culture Media | factor:empty vector	RAW_FILE_NAME(raw file name)=MR89-11
SUBJECT_SAMPLE_FACTORS           	-	SUM159-EV-3M	Sample source:Culture Media | factor:empty vector	RAW_FILE_NAME(raw file name)=MR89-12
#COLLECTION
CO:COLLECTION_SUMMARY            	At the end of the cell culturing timecourse, the media were harvested and
CO:COLLECTION_SUMMARY            	centrifuged at 1500 rpm, 4℃. The resulting supernatants were collected and
CO:COLLECTION_SUMMARY            	frozen at -80 ℃.
CO:SAMPLE_TYPE                   	Culture Media
#TREATMENT
TR:TREATMENT_SUMMARY             	TNBC cell line SUM159 with stable TDO2 overexpression (TDO2 OE) were cultured
TR:TREATMENT_SUMMARY             	relative to empty vector (EV). Culture media was not changed during the duration
TR:TREATMENT_SUMMARY             	of the experiments.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Supernatant aliquots were thawed on ice then 20 uL was treated with 480 uL of
SP:SAMPLEPREP_SUMMARY            	ice cold 5:3:2 methanol:acetonitrile:water (v/v/v). Samples were vortexed 30 min
SP:SAMPLEPREP_SUMMARY            	at 4 degrees C followed by centrifugation for 10 min at 18,000 g. Aliquots of
SP:SAMPLEPREP_SUMMARY            	supernatant (50 uL) were dried using a speedvac then reconstituted in an
SP:SAMPLEPREP_SUMMARY            	equivalent volume of 0.1% formic acid. Samples were maintained at 4°C until
SP:SAMPLEPREP_SUMMARY            	analysis that same day.
SP:PROCESSING_STORAGE_CONDITIONS 	4℃
SP:EXTRACT_STORAGE               	-80℃
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Positive C18
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Vanquish
CH:COLUMN_NAME                   	Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
CH:SOLVENT_A                     	100% water; 0.1% formic acid
CH:SOLVENT_B                     	100% acetonitrile; 0.1% formic acid
CH:FLOW_GRADIENT                 	0-0.5 min 5% B, 0.5-1.1 min 5-95% B, 1.1-2.75 min hold at 95% B, 2.75-3 min
CH:FLOW_GRADIENT                 	95-5% B, 3-5 min hold at 5% B
CH:FLOW_RATE                     	450 uL/min
CH:COLUMN_TEMPERATURE            	45
CH:SAMPLE_INJECTION              	6 uL
#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                   	Resolution 70,000, scan range 65-900 m/z, maximum injection time 200 ms,
MS:MS_COMMENTS                   	microscans 2, automatic gain control (AGC) 3 x 10^6 ions, source voltage 4.0 kV,
MS:MS_COMMENTS                   	capillary temperature 320 C, and sheath gas 45, auxiliary gas 15, and sweep gas
MS:MS_COMMENTS                   	0 (all nitrogen). Data converted to mzXML using RawConverter. Metabolites were
MS:MS_COMMENTS                   	annotated and integrated using Maven in conjunction with the KEGG database.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	peak area
MS_METABOLITE_DATA_START
Samples	SUM159-EV-1M	SUM159-EV-2M	SUM159-EV-3M	SUM159-TDO2-1M	SUM159-TDO2-2M	SUM159-TDO2-3M
Factors	Sample source:Culture Media | factor:empty vector	Sample source:Culture Media | factor:empty vector	Sample source:Culture Media | factor:empty vector	Sample source:Culture Media | factor:TDO2 overexpression	Sample source:Culture Media | factor:TDO2 overexpression	Sample source:Culture Media | factor:TDO2 overexpression
L-Tryptophan	28546270	23783830	31727850	14958320	18890990	17979380
L-Formylkynurenine (tentative)	46634	52011	48232	149789	165401	165494
L-Kynurenine	459684	429486	479309	2066250	2868560	2506739
Anthranilate	351974	392082	417591	279934	331000	306891
Indole (tentative)	466662	465486	517499	473952	542842	417968
5-Hydroxyindoleacetate	116567	141841	133041	575797	794420	614933
3-Methyldioxyindole (tentative)	34032	43501	48855	57618	58320	59378
Indole-3-acetaldehyde	352505	328464	329811	590662	568893	470054
Indole-3-acetate	59911	59034	58567	19823	25824	21674
Indolepyruvate	896686	574198	451713	332812	324162	400223
6-Hydroxykynurenic acid	517166	680776	760742	337852	274984	287868
L-Tryptophanamide	83013	59135	62509	20781	20782	21825
Pyridine-2-3-dicarboxylate	16916	10371	0	11716	10212	10085
Picolinic acid	4555550	4203408	5110572	3904299	4009088	3841055
(Z)-5-Oxohex-2-enedioate	381428	378640	336294	112061	73782	124840
2-Aminomuconate	444255	333430	296293	251947	284118	263271
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	m/z	RT	KEGG ID
L-Tryptophan	205.097	1.679	C00078
L-Formylkynurenine (tentative)	237.0865	1.456	C02700
L-Kynurenine	209.0918	1.391	C00328
Anthranilate	138.0533	0.655	C00108
Indole (tentative)	118.065	1.743	C00463
5-Hydroxyindoleacetate	192.0641	1.396	C05635
3-Methyldioxyindole (tentative)	164.0704	0.802	C05834
Indole-3-acetaldehyde	160.0756	1.127	C00637
Indole-3-acetate	176.0707	1.709	C00954
Indolepyruvate	204.0632	0.891	C00331
6-Hydroxykynurenic acid	206.0478	0.880	C08480
L-Tryptophanamide	204.1123	1.680	C00977
Pyridine-2-3-dicarboxylate	168.0323	0.762	C03722
Picolinic acid	124.0409	0.556	C10164
(Z)-5-Oxohex-2-enedioate	159.0279	0.878	C03453
2-Aminomuconate	158.045	0.875	C02220
METABOLITES_END
#END