#METABOLOMICS WORKBENCH juliehaines_20240917_112835 DATATRACK_ID:5202 STUDY_ID:ST003488 ANALYSIS_ID:AN005726 PROJECT_ID:PR002140 VERSION 1 CREATED_ON September 17, 2024, 1:22 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 metabolite profiling of cell supernatants (i.e. cell culture media) ST:STUDY_TITLE from TNBC cell line BT549 grown in suspension with TDO2 knockdown and IDO1 ST:STUDY_TITLE compensation. ST:STUDY_SUMMARY Conditioned media harvested from TNBC cell line BT549 with stable TDO2 knockdown ST:STUDY_SUMMARY (shTDO2) but harbor IDO1 compensation. The cells were treated with TDO2/IDO1 ST:STUDY_SUMMARY dual inhibitor AT-0174, IDO1 inhibitor epacadostat, or TDO2 inhibitor 680C91 for ST:STUDY_SUMMARY 48 hours. 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 - AA22-2M-1 treatment:DMSO | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA22-2M-1_r12+ SUBJECT_SAMPLE_FACTORS - AA22-2M-2 treatment:DMSO | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA22-2M-2_r4+ SUBJECT_SAMPLE_FACTORS - AA22-2M-3 treatment:DMSO | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA22-2M-3_r9+ SUBJECT_SAMPLE_FACTORS - AA22-2M-4 treatment:10uM AT0174 | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA22-2M-4_r1+ SUBJECT_SAMPLE_FACTORS - AA22-2M-5 treatment:10uM AT0174 | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA22-2M-5_r6+ SUBJECT_SAMPLE_FACTORS - AA22-2M-6 treatment:10uM AT0174 | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA22-2M-6_r7+ SUBJECT_SAMPLE_FACTORS - AA22-2M-7 treatment:10uM 680c91 | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA22-2M-7_r10+ SUBJECT_SAMPLE_FACTORS - AA22-2M-8 treatment:10uM 680c91 | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA22-2M-8_r8+ SUBJECT_SAMPLE_FACTORS - AA22-2M-9 treatment:10uM 680c91 | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA22-2M-9_r11+ SUBJECT_SAMPLE_FACTORS - AA22-2M-10 treatment:10uM Epacadotstat | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA22-2M-10_r5+ SUBJECT_SAMPLE_FACTORS - AA22-2M-11 treatment:10uM Epacadotstat | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA22-2M-11_r3+ SUBJECT_SAMPLE_FACTORS - AA22-2M-12 treatment:10uM Epacadotstat | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA22-2M-12_r2+ SUBJECT_SAMPLE_FACTORS - AA22-3M-1 treatment:shSCR | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA22-3M-1_r10+ SUBJECT_SAMPLE_FACTORS - AA22-3M-2 treatment:shSCR | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA22-3M-2_r7+ SUBJECT_SAMPLE_FACTORS - AA22-3M-3 treatment:shSCR | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA22-3M-3_r5+ SUBJECT_SAMPLE_FACTORS - AA22-3M-4 treatment:shTDO2-082 | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA22-3M-4_r8+ SUBJECT_SAMPLE_FACTORS - AA22-3M-5 treatment:shTDO2-082 | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA22-3M-5_r3+ SUBJECT_SAMPLE_FACTORS - AA22-3M-6 treatment:shTDO2-082 | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA22-3M-6_r6+ SUBJECT_SAMPLE_FACTORS - AA22-3M-7 treatment:shTDO2-098 | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA22-3M-7_r1+ SUBJECT_SAMPLE_FACTORS - AA22-3M-8 treatment:shTDO2-098 | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA22-3M-8_r2+ SUBJECT_SAMPLE_FACTORS - AA22-3M-9 treatment:shTDO2-098 | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA22-3M-9_r11+ SUBJECT_SAMPLE_FACTORS - AA22-3M-10 treatment:shTDO2-054 | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA22-3M-10_r12+ SUBJECT_SAMPLE_FACTORS - AA22-3M-11 treatment:shTDO2-054 | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA22-3M-11_r9+ SUBJECT_SAMPLE_FACTORS - AA22-3M-12 treatment:shTDO2-054 | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA22-3M-12_r4+ SUBJECT_SAMPLE_FACTORS - AA22-4M-1 treatment:EV | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA22-4M-1_r2+ SUBJECT_SAMPLE_FACTORS - AA22-4M-2 treatment:EV | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA22-4M-2_r6+ SUBJECT_SAMPLE_FACTORS - AA22-4M-3 treatment:EV | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA22-4M-3_r3+ SUBJECT_SAMPLE_FACTORS - AA22-4M-4 treatment:TDO2 OE | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA22-4M-4_r4+ SUBJECT_SAMPLE_FACTORS - AA22-4M-5 treatment:TDO2 OE | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA22-4M-5_r1+ SUBJECT_SAMPLE_FACTORS - AA22-4M-6 treatment:TDO2 OE | Sample source:conditioned media RAW_FILE_NAME(raw file name)=AA22-4M-6_r5+ #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 Breast cancer cells #TREATMENT TR:TREATMENT_SUMMARY TNBC cell line BT549 with stable TDO2 knockdown (shTDO2) was first cultured for TR:TREATMENT_SUMMARY 24 hours and treated with DMSO (control), 10 µM AT-0174 and 10 µM 680c91 or 10 TR:TREATMENT_SUMMARY µM Epacadostat for 48 hours. For comparison, BT549 with stable TDO2 TR:TREATMENT_SUMMARY overexpression (TDO2 OE) were also cultured relative to empty vector (EV). TR:TREATMENT_SUMMARY Culture media was not changed during the duration 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 AA22-2M-1 AA22-2M-2 AA22-2M-3 AA22-2M-4 AA22-2M-5 AA22-2M-6 AA22-2M-7 AA22-2M-8 AA22-2M-9 AA22-2M-10 AA22-2M-11 AA22-2M-12 AA22-3M-1 AA22-3M-2 AA22-3M-3 AA22-3M-4 AA22-3M-5 AA22-3M-6 AA22-3M-7 AA22-3M-8 AA22-3M-9 AA22-3M-10 AA22-3M-11 AA22-3M-12 AA22-4M-1 AA22-4M-2 AA22-4M-3 AA22-4M-4 AA22-4M-5 AA22-4M-6 Factors treatment:DMSO | Sample source:conditioned media treatment:DMSO | Sample source:conditioned media treatment:DMSO | 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 680c91 | Sample source:conditioned media treatment:10uM 680c91 | Sample source:conditioned media treatment:10uM 680c91 | Sample source:conditioned media treatment:10uM Epacadotstat | Sample source:conditioned media treatment:10uM Epacadotstat | Sample source:conditioned media treatment:10uM Epacadotstat | Sample source:conditioned media treatment:shSCR | Sample source:conditioned media treatment:shSCR | Sample source:conditioned media treatment:shSCR | Sample source:conditioned media treatment:shTDO2-082 | Sample source:conditioned media treatment:shTDO2-082 | Sample source:conditioned media treatment:shTDO2-082 | Sample source:conditioned media treatment:shTDO2-098 | Sample source:conditioned media treatment:shTDO2-098 | Sample source:conditioned media treatment:shTDO2-098 | Sample source:conditioned media treatment:shTDO2-054 | Sample source:conditioned media treatment:shTDO2-054 | Sample source:conditioned media treatment:shTDO2-054 | Sample source:conditioned media treatment:EV | Sample source:conditioned media treatment:EV | Sample source:conditioned media treatment:EV | Sample source:conditioned media treatment:TDO2 OE | Sample source:conditioned media treatment:TDO2 OE | Sample source:conditioned media treatment:TDO2 OE | Sample source:conditioned media L-tryptophan 29039746 31019402 51691920 40949092 49961472 46398556 57266652 54274224 57713484 43295624 41742596 35434896 25051312 31835658 39025512 27325466 35133772 40448164 46905996 34624668 36421892 51047380 53893468 45344544 14722827 14518777 16573717 15627416 14916571 15783655 5-Hydroxyindoleacetate 170023 171926 216435 139105 217089 160567 321461 264536 350343 208940 128202 104934 141441 194580 261404 109039 140020 155413 312393 171088 339742 130003 146563 100451 43517 39727 39773 106783 93812 94386 3-Methyleneoxindole 2129961 2232913 3603075 2722792 2586069 3412304 3970109 4067519 4033460 3288453 2953398 2515269 1739902 2344542 2775596 1857259 2419900 2792216 3120773 2381029 3319676 3344828 2896378 3214764 1177169 1126172 1255256 1195154 1101266 1199180 Indole 1044800 1072472 1759974 1211864 1157037 1607663 1851806 1991941 1935567 1622847 1363710 1224275 826161 1151159 1258312 934824 1090373 1351673 1436622 1094904 1657047 1476406 1550356 1586468 513619 617500 577921 554817 485041 533595 Indole-3-acetaldehyde 2161603 2584748 2787298 3651278 3976026 2929557 3191039 2906029 3643875 3063516 2698315 2605311 1996373 2720718 3376723 575851 801207 998352 2616627 1857277 1729114 928434 741104 712065 73723 79479 90328 108269 90581 99867 Indole-3-acetate 133505 138685 189281 147983 209567 210340 173897 145916 229829 158871 134587 106273 112930 179320 171316 72303 80261 86160 129124 113188 112107 73301 71010 76751 15394 15602 17326 18291 18574 23598 Indolepyruvate 1078314 1177797 1517649 1369953 1368696 1317060 1455585 1593092 1724840 1427805 1272495 1271817 761531 1101640 1236754 1132268 1248923 1375914 1502525 1142180 1618884 1642810 1396264 1330304 675270 778045 787832 707864 884004 711374 Indoxyl 806318 940751 1267885 943135 1234179 1325710 1078596 1255155 1805968 1322222 1223321 1128671 765968 649641 1163530 953193 1016573 1121488 1233311 1074688 997870 937994 1049259 861936 308529 285657 429344 454758 494857 422762 kynurenine 483107 558640 729384 438960 664004 485093 802979 837109 1074548 607768 439021 353264 471777 561706 824306 350452 410186 510820 1027744 645027 1020693 433390 396329 342933 132982 114666 143811 320830 289002 321327 N-formyl kynurenine 177437 210931 350811 118234 152108 142016 272459 224426 321318 238737 131465 118108 111532 169812 247851 69802 78024 108327 404134 262453 343889 117877 116660 105869 21322 17617 24353 93617 83879 85241 Anthranilate 5712076 4878580 7970116 6948936 7198687 7510431 9282137 10147461 11036409 7243445 6802807 5905130 351232 542877 591030 236853 232505 333382 3285473 1583431 2680397 10996969 10351368 9858417 5042326 4825960 5511180 5639728 5048863 5449201 Picolinic acid 3450030 3668861 5578138 3986421 5127903 4829064 7334105 6637243 8242587 4347002 2947299 3174870 5208120 3768803 7558568 3072468 3202641 4282490 11614952 8737217 8937214 728629 700063 683066 56303 51731 40158 55883 60418 54809 g-Oxalo-crotonate 4986046 11661549 15364411 8581767 9067565 14933724 10951351 16269353 22057952 14251443 14086815 13892461 329550 257516 198419 1355343 1214882 1651303 672551 795411 990633 424818 425923 589445 23836 12865 17424 18892 17612 25047 Hydroxyindole-acetylglycine 20470 15556 28917 12168 18213 18973 24784 52437 23410 27190 18244 22458 11786 19975 18984 15561 16105 21002 15418 16662 17960 37806 34616 37317 12162 10824 8729 6484 6827 4858 8-Methoxykynurenate 8507 4609 16518 14538 7407 5005 13995 13747 16069 11168 5043 11291 33989 25117 39922 39408 67419 46089 59420 59494 29478 83305 79209 75484 9767 8085 6977 3499 5836 4711 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID m/z RT L-tryptophan C00078 205.0966 1.7640 5-Hydroxyindoleacetate C05635 192.064774 1.541 3-Methyleneoxindole C02796 146.059662 1.772 Indole C00463 118.065163 1.775 Indole-3-acetaldehyde C00637 160.075226 1.328 Indole-3-acetate C00954 176.070724 1.872 Indolepyruvate C00331 204.0625 0.992 Indoxyl C05658 134.059647 0.956 kynurenine C00328 209.091217 1.541 N-formyl kynurenine C02700 237.086395 1.638 Anthranilate C00108 138.054581 1.802 Picolinic acid C10164 124.039146 0.954 g-Oxalo-crotonate C03453 159.030365 0.972 Hydroxyindole-acetylglycine C05832 249.086304 1.769 8-Methoxykynurenate C05830 220.06131 1.855 METABOLITES_END #END