#METABOLOMICS WORKBENCH juliehaines_20240917_092840 DATATRACK_ID:5201 STUDY_ID:ST003487 ANALYSIS_ID:AN005725 PROJECT_ID:PR002140 VERSION 1 CREATED_ON September 18, 2024, 8:54 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 TNBC cell line BT549 grown in suspension with ST:STUDY_TITLE TDO inhibition. ST:STUDY_SUMMARY Indole-focused metabolomics analysis of TNBC cells (BT549) with pharmacological ST:STUDY_SUMMARY inhibition of TDO2 by AT-0174 or 680c91. 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 Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 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 - BTS-68048-1 treatment:10 uM 680c91 | Sample source:Breast cancer cells RAW_FILE_NAME=DS1-30-01+ SUBJECT_SAMPLE_FACTORS - BTS-68048-2 treatment:10 uM 680c91 | Sample source:Breast cancer cells RAW_FILE_NAME=DS1-30-02+ SUBJECT_SAMPLE_FACTORS - BTS-68048-3 treatment:10 uM 680c91 | Sample source:Breast cancer cells RAW_FILE_NAME=DS1-30-03+ SUBJECT_SAMPLE_FACTORS - BTS-10A48-1 treatment:10 uM AT0174 | Sample source:Breast cancer cells RAW_FILE_NAME=DS1-30-04+ SUBJECT_SAMPLE_FACTORS - BTS-10A48-2 treatment:10 uM AT0174 | Sample source:Breast cancer cells RAW_FILE_NAME=DS1-30-05+ SUBJECT_SAMPLE_FACTORS - BTS-10A48-3 treatment:10 uM AT0174 | Sample source:Breast cancer cells RAW_FILE_NAME=DS1-30-06+ SUBJECT_SAMPLE_FACTORS - BTS-1A48-1 treatment:1 uM AT0174 | Sample source:Breast cancer cells RAW_FILE_NAME=DS1-30-07+ SUBJECT_SAMPLE_FACTORS - BTS-1A48-2 treatment:1 uM AT0174 | Sample source:Breast cancer cells RAW_FILE_NAME=DS1-30-08+ SUBJECT_SAMPLE_FACTORS - BTS-1A48-3 treatment:1 uM AT0174 | Sample source:Breast cancer cells RAW_FILE_NAME=DS1-30-09+ SUBJECT_SAMPLE_FACTORS - BTS-D48-1 treatment:DMSO vehicle | Sample source:Breast cancer cells RAW_FILE_NAME=DS1-30-10+ SUBJECT_SAMPLE_FACTORS - BTS-D48-2 treatment:DMSO vehicle | Sample source:Breast cancer cells RAW_FILE_NAME=DS1-30-11+ SUBJECT_SAMPLE_FACTORS - BTS-D48-3 treatment:DMSO vehicle | Sample source:Breast cancer cells RAW_FILE_NAME=DS1-30-12+ #COLLECTION CO:COLLECTION_SUMMARY At the end of the cell culturing timecourse, the media were removed then the CO:COLLECTION_SUMMARY cells were detached using 0.5% trypsin/EDTA and then centrifuged at 1500 rpm for CO:COLLECTION_SUMMARY 5 mins at 4℃, and the supernatants were discarded. Next, PBS was used to CO:COLLECTION_SUMMARY resuspend the cell pellets and centrifuged again at 13000 rpm for 15 mins at CO:COLLECTION_SUMMARY 4℃. The supernatant then discarded. The cell pellets were frozen at -80℃ CO:COLLECTION_SUMMARY until analysis. CO:SAMPLE_TYPE Breast cancer cells #TREATMENT TR:TREATMENT_SUMMARY TNBC cell line BT549 was cultured in suspension for 24 hours then treated with TR:TREATMENT_SUMMARY DMSO (control), 1 µM AT-0174, 10 µM AT-0174, or 10 uM 680c91 for 48 hours. TR:TREATMENT_SUMMARY Culture media was not changed during the duration of the experiments. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites were extracted from frozen cell pellets using ice cold 5:3:2 SP:SAMPLEPREP_SUMMARY methanol:acetonitrile:water (v/v/v) at 2 million cells per mL. Samples were SP:SAMPLEPREP_SUMMARY vortexed 30 min at 4 degrees C followed by centrifugation for 10 min at 18,000 SP:SAMPLEPREP_SUMMARY g. Aliquots of supernatant (50 uL) were dried using a speedvac then SP:SAMPLEPREP_SUMMARY reconstituted in an equivalent volume of 0.1% formic acid. Samples were SP:SAMPLEPREP_SUMMARY maintained at 4°C until 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 BTS-68048-1 BTS-68048-2 BTS-68048-3 BTS-10A48-1 BTS-10A48-2 BTS-10A48-3 BTS-1A48-1 BTS-1A48-2 BTS-1A48-3 BTS-D48-1 BTS-D48-2 BTS-D48-3 Factors treatment:10 uM 680c91 | Sample source:Breast cancer cells treatment:10 uM 680c91 | Sample source:Breast cancer cells treatment:10 uM 680c91 | Sample source:Breast cancer cells treatment:10 uM AT0174 | Sample source:Breast cancer cells treatment:10 uM AT0174 | Sample source:Breast cancer cells treatment:10 uM AT0174 | Sample source:Breast cancer cells treatment:1 uM AT0174 | Sample source:Breast cancer cells treatment:1 uM AT0174 | Sample source:Breast cancer cells treatment:1 uM AT0174 | Sample source:Breast cancer cells treatment:DMSO vehicle | Sample source:Breast cancer cells treatment:DMSO vehicle | Sample source:Breast cancer cells treatment:DMSO vehicle | Sample source:Breast cancer cells 5-Hydroxyindoleacetate 1303946 1186771 1385611 1532727 2068595 2234090 2239447 1575651 1864273 1103217 1665920 1914503 3-Methyleneoxindole 269476.2 208763.2 211415 341066.7 431094.9 481003.6 555281.4 358511 378021.4 235818.6 278904.6 204984.7 Indole 173859.9 126727.4 122866 207328.7 243392.9 247850.6 304927.6 192168.3 221076.9 124723.4 161549.9 137579.2 Indole-3-acetaldehyde 12962.35 15729.44 12636.06 23491.9 30092.78 30116.31 36467.04 19347.64 20085.52 15679.97 14453.99 11660.11 Indolepyruvate 31808.16 20671.26 41705.55 18355.78 17969.62 10158.23 20622.97 19751.67 28679.53 23458.13 25553.05 99479.82 6-Hydroxykynurenic acid 157748.7 114814.9 109036.5 199756.7 326943.1 319254.2 214781 311686.1 458080.8 201679.4 268490.2 122377.9 kynurenine 283932.7 181495.9 174208 609934.8 817953 987054.2 2187093 1261708 1898506 2887910 3896185 2288864 N-formyl kynurenine 152752 144395.7 168986.7 535601.4 648561.3 837486 1507285 942777.9 1231727 1993090 1994012 1212064 Anthranilate 103755.3 162547.9 129255 103008.2 159125.2 126821.8 153518.7 119581.1 119524 100215.1 97350.18 110354.2 Picolinic acid 1622467 1772881 1905941 1949704 2746598 2692841 2950049 2486492 2496974 1936893 2347040 2316351 g-Oxalo-crotonate 104202.4 106104.7 194716.4 145186 97252.78 161382 102502.1 112501.8 107544.9 124265.9 121960.8 236271.8 2-Aminomuconate 284481.8 371685 400550.2 264129 340927 326209.4 236585.7 337216.8 379533.2 269264 350335.2 269853.8 Formyl-5-hydroxy-kynurenamine 283932.7 181495.9 174208 609934.8 817953 987054.2 2187093 1261708 1898506 2887910 3896185 2288864 Hydroxyindole-acetylglycine 1262.626 0 0 2357.488 7871.647 4234.245 7997.068 2191.733 2559.687 3288.76 2845.748 1346.52 Kynurenate 31943.64 27777.16 25528 27963.37 34178.72 38678.75 30192.2 31415.58 33563.79 25128.8 26527.53 29343.83 Tryptamine 48111.45 37568.44 40874.37 50538.03 105767.2 81712.32 74532.44 63002.58 80077.44 63353.29 70291.28 37139.21 5-hydroxytryptophan 25984.74 28108.28 23348.87 26491.33 41973.21 43130.95 44466.79 31389.71 37393.4 38258.78 37873.84 24083.88 L-tryptophan 15677400 11302460 11723770 20300520 22960290 28243820 29232070 19477080 21839530 13007890 15121800 11626150 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID m/z RT 5-Hydroxyindoleacetate C05635 192.0618 0.5667723 3-Methyleneoxindole C02796 146.0603 1.742382 Indole C00463 118.0656 1.748629 Indole-3-acetaldehyde C00637 160.0761 1.733175 Indolepyruvate C00331 204.0636 0.877594 6-Hydroxykynurenic acid C08480 206.0484 0.8706 kynurenine C00328 209.0922 1.337228 N-formyl kynurenine C02700 237.0872 1.390947 Anthranilate C00108 138.0552 1.757466 Picolinic acid C10164 124.0402 0.5641276 g-Oxalo-crotonate C03453 159.03 0.8567833 2-Aminomuconate C02220 158.0434 0.8271907 Formyl-5-hydroxy-kynurenamine C05647 209.0922 1.325983 Hydroxyindole-acetylglycine C05832 249.085 1.591549 Kynurenate C01717 190.0499 0.9183065 Tryptamine C00398 161.1075 1.756924 5-hydroxytryptophan C00643 221.0923 1.048222 L-tryptophan C00078 205.0975 1.736209 METABOLITES_END #END