#METABOLOMICS WORKBENCH juliehaines_20240918_063241 DATATRACK_ID:5204 STUDY_ID:ST003489 ANALYSIS_ID:AN005728 PROJECT_ID:PR002140 VERSION 1 CREATED_ON September 18, 2024, 7:13 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 Core director Angelo D'Alessandro, study PI 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 pathway profiling of conditioned media from TNBC cell line MDA-MB-231 ST:STUDY_TITLE +/- inhibitors of the TDO/IDO pathway. ST:STUDY_SUMMARY Conditioned media harvested from TNBC cell line MDA-MB-231 with and without ST:STUDY_SUMMARY treatment with TDO2/IDO1 dual inhibitor AT-0174 and IDO1 inhibitor epacadostat. 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 - AA90-M231-1 treatment:Vehicle | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA90-M231-1 SUBJECT_SAMPLE_FACTORS - AA90-M231-2 treatment:Vehicle | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA90-M231-2 SUBJECT_SAMPLE_FACTORS - AA90-M231-3 treatment:Vehicle | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA90-M231-3 SUBJECT_SAMPLE_FACTORS - AA90-M231-4 treatment:10uM AT0174 | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA90-M231-4 SUBJECT_SAMPLE_FACTORS - AA90-M231-5 treatment:10uM AT0174 | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA90-M231-5 SUBJECT_SAMPLE_FACTORS - AA90-M231-6 treatment:10uM AT0174 | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA90-M231-6 SUBJECT_SAMPLE_FACTORS - AA90-M231-7 treatment:10uM Epacadostat | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA90-M231-7 SUBJECT_SAMPLE_FACTORS - AA90-M231-8 treatment:10uM Epacadostat | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA90-M231-8 SUBJECT_SAMPLE_FACTORS - AA90-M231-9 treatment:10uM Epacadostat | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA90-M231-9 #COLLECTION CO:COLLECTION_SUMMARY At the end of the experimental timecourse, media were harvested and centrifuged CO:COLLECTION_SUMMARY at 1500 rpm, 4℃. The supernatants were collected and frozen at -80℃ prior to CO:COLLECTION_SUMMARY metabolomics analysis. CO:SAMPLE_TYPE Culture Media #TREATMENT TR:TREATMENT_SUMMARY The TNBC cell line MDA-MB-231 was cultured for 24 hours then treated with DMSO TR:TREATMENT_SUMMARY (control), 10 µM AT-0174 or 10 µM epacadostat for 48 hours. Culture media was TR:TREATMENT_SUMMARY not changed during the duration of the experiments. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Media aliquots were thawed on ice then metabolites extracted from a 10 uL SP:SAMPLEPREP_SUMMARY aliquot using 240 uL of cold 5:3:2 MeOH:acetonitrile:water. Samples were SP:SAMPLEPREP_SUMMARY vortexed 30 min at 4 degrees C then supernatants clarified by centrifugation (10 SP:SAMPLEPREP_SUMMARY min, 10,000 g, 4 degrees C). A 150 uL of supernatant was dried using a speedvac SP:SAMPLEPREP_SUMMARY then resuspended in 150 uL of 0.1% formic acid and transferred to autosampler SP:SAMPLEPREP_SUMMARY vials. 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 10 uL #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Orbitrap Exploris 120 MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS We use a Thermo Orbitrap Exploris 120. Resolution 120,000, scan range 65-975 MS:MS_COMMENTS m/z, maximum injection time 100 ms, microscans 1, automatic gain control (AGC) MS:MS_COMMENTS detection duration 20 msec, source voltage 3.5 kV, capillary temperature 320 C, MS:MS_COMMENTS vaporizer temp 200 C, and sheath gas 50, auxiliary gas 10, and sweep gas 1 (all MS:MS_COMMENTS 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 AA90-M231-1 AA90-M231-2 AA90-M231-3 AA90-M231-4 AA90-M231-5 AA90-M231-6 AA90-M231-7 AA90-M231-8 AA90-M231-9 Factors treatment:Vehicle | Sample source:conditioned media treatment:Vehicle | Sample source:conditioned media treatment:Vehicle | Sample source:conditioned media treatment:10uM AT0174 | Sample source:conditioned media treatment:10uM AT0174 | Sample source:conditioned media treatment:10uM AT0174 | Sample source:conditioned media treatment:10uM Epacadostat | Sample source:conditioned media treatment:10uM Epacadostat | Sample source:conditioned media treatment:10uM Epacadostat | Sample source:conditioned media L-tryptophan 31292354 31103488 33853740 58413684 67764552 66651572 30845518 25539704 22852898 5-Hydroxyindoleacetate 8356312 9219125 9943541 6359090 7236370 7932280 10445584 8945125 9311665 3-Methyleneoxindole 308098 320594 326180 624078 688217 598747 287896 268434 239343 Indole-3-acetaldehyde 927350 894774 1011945 860051 946350 934462 976407 833005 764581 Indole-3-acetate 11844325 10881296 12814083 10128045 13371160 12938152 12242081 9549732 8347080 Indoxyl 712127 748455 706803 714810 758057 882641 790029 653086 661689 5-hydroxytryptophan 809606 778747 926784 692409 856075 830430 920578 685033 677585 kynurenine 36824784 38962892 41959956 28586208 31733554 30490298 43555296 38784884 36420048 N-formyl kynurenine 3868778 3879298 4466581 2688372 3035013 2997742 4172619 3416164 3093764 Anthranilate 21055676 24237514 25260006 18662134 19611090 19940924 26898814 22919814 20083426 2-Aminomuconate 123326 73409 89276 54844 152109 50335 94379 79649 60280 Kynurenate 250586 248224 288567 223798 230473 212341 278782 229880 213129 8-Methoxykynurenate 103483 162124 98213 81539 75535 76464 91254 194082 91536 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID m/z RT L-tryptophan C00078 205.0971 1.7170 5-Hydroxyindoleacetate C05635 192.06546 1.355 3-Methyleneoxindole C02796 146.060074 1.713 Indole-3-acetaldehyde C00637 160.075684 1.211 Indole-3-acetate C00954 176.070587 1.916 Indoxyl C05658 134.060104 0.824 5-hydroxytryptophan C00643 221.092102 1.022 kynurenine C00328 209.092056 1.352 N-formyl kynurenine C02700 237.086975 1.41 Anthranilate C00108 138.055023 1.855 2-Aminomuconate C02220 158.044754 0.823 Kynurenate C01717 190.049835 1.735 8-Methoxykynurenate C05830 220.06041 1.853 METABOLITES_END #END