#METABOLOMICS WORKBENCH juliehaines_20240917_091515 DATATRACK_ID:5200 STUDY_ID:ST003486 ANALYSIS_ID:AN005724 PROJECT_ID:PR002140 VERSION 1 CREATED_ON September 18, 2024, 8:45 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 (culture media) from TNBC ST:STUDY_TITLE cell line BT549 grown in suspension with TDO inhibition. ST:STUDY_SUMMARY Indole-focused metabolomics analysis of cell supernatants (i.e. cell culture ST:STUDY_SUMMARY media) from TNBC cell lines with pharmacological inhibition of TDO2 by AT-0174 ST:STUDY_SUMMARY 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-68048-1M treatment:10 uM 680c91 | Sample source:conditioned media RAW_FILE_NAME(raw file name)=DS1-30-13+ SUBJECT_SAMPLE_FACTORS - BTS-68048-2M treatment:10 uM 680c91 | Sample source:conditioned media RAW_FILE_NAME(raw file name)=DS1-30-14+ SUBJECT_SAMPLE_FACTORS - BTS-68048-3M treatment:10 uM 680c91 | Sample source:conditioned media RAW_FILE_NAME(raw file name)=DS1-30-15+ SUBJECT_SAMPLE_FACTORS - BTS-10A48-1M treatment:10 uM AT0174 | Sample source:conditioned media RAW_FILE_NAME(raw file name)=DS1-30-16+ SUBJECT_SAMPLE_FACTORS - BTS-10A48-2M treatment:10 uM AT0174 | Sample source:conditioned media RAW_FILE_NAME(raw file name)=DS1-30-17+ SUBJECT_SAMPLE_FACTORS - BTS-10A48-3M treatment:10 uM AT0174 | Sample source:conditioned media RAW_FILE_NAME(raw file name)=DS1-30-18+ SUBJECT_SAMPLE_FACTORS - BTS-1A48-1M treatment:1 uM AT0174 | Sample source:conditioned media RAW_FILE_NAME(raw file name)=DS1-30-19+ SUBJECT_SAMPLE_FACTORS - BTS-1A48-2M treatment:1 uM AT0174 | Sample source:conditioned media RAW_FILE_NAME(raw file name)=DS1-30-20+ SUBJECT_SAMPLE_FACTORS - BTS-1A48-3M treatment:1 uM AT0174 | Sample source:conditioned media RAW_FILE_NAME(raw file name)=DS1-30-21+ SUBJECT_SAMPLE_FACTORS - BTS-D48-1M treatment:DMSO vehicle | Sample source:conditioned media RAW_FILE_NAME(raw file name)=DS1-30-22+ SUBJECT_SAMPLE_FACTORS - BTS-D48-2M treatment:DMSO vehicle | Sample source:conditioned media RAW_FILE_NAME(raw file name)=DS1-30-23+ SUBJECT_SAMPLE_FACTORS - BTS-D48-3M treatment:DMSO vehicle | Sample source:conditioned media RAW_FILE_NAME(raw file name)=DS1-30-24+ #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 BT549 was cultured for 24 hours then treated with DMSO (control), TR:TREATMENT_SUMMARY 1 µM AT-0174, 10 µM AT-0174, or 10 uM 680c91 for 48 hours. Culture media was TR:TREATMENT_SUMMARY 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 BTS-68048-1M BTS-68048-2M BTS-68048-3M BTS-10A48-1M BTS-10A48-2M BTS-10A48-3M BTS-1A48-1M BTS-1A48-2M BTS-1A48-3M BTS-D48-1M BTS-D48-2M BTS-D48-3M Factors treatment:10 uM 680c91 | Sample source:conditioned media treatment:10 uM 680c91 | Sample source:conditioned media treatment:10 uM 680c91 | Sample source:conditioned media treatment:10 uM AT0174 | Sample source:conditioned media treatment:10 uM AT0174 | Sample source:conditioned media treatment:10 uM AT0174 | Sample source:conditioned media treatment:1 uM AT0174 | Sample source:conditioned media treatment:1 uM AT0174 | Sample source:conditioned media treatment:1 uM AT0174 | Sample source:conditioned media treatment:DMSO vehicle | Sample source:conditioned media treatment:DMSO vehicle | Sample source:conditioned media treatment:DMSO vehicle | Sample source:conditioned media 5-Hydroxyindoleacetate 4782790 5522946 4656318 5238430 4643834 3895843 4909326 4539077 4807516 5368992 3861478 5009230 3-methyldioxyindole 35894.18 79072.48 44826.86 134354.3 46453.96 23426.96 128968.9 53579.77 137227.4 33605.43 33889.6 30887.63 3-Methyleneoxindole 1171177 1458937 1625569 1818440 1382399 1877524 1767991 1344006 1462200 1036507 1290743 669262.5 Indole 595884.5 808628.4 848601.8 939298.8 774896.8 985219.2 991921.5 741315.7 719764.8 602300.9 746742.1 349614.6 Indole-3-acetaldehyde 60346.61 109900.9 106981 95176.19 64155.45 112182.6 97425.27 107252.5 68961.32 76529.27 110541.2 27602.64 Indole-3-acetate 88076.13 8585.562 13814.44 170058.1 7822.836 4427.31 151384.2 109205.9 136063.5 167108.5 16548.61 119299.8 Indolepyruvate 138920.7 354823.6 245524.2 117093.4 155083.2 195729.6 140696.1 192602.4 189127 212597.1 137944.5 190921 Indoxyl 187043.6 314643.3 301213.5 292693.5 196120.3 278353.9 303171.1 200493.2 246457.2 258711.1 248473.2 142128.8 kynurenine 4303696 1624258 2353268 5552108 4612482 5666338 6346780 5389814 6029606 10600000 13200000 7945914 N-formyl kynurenine 199256.7 64711.69 101129 213784.1 306485.2 278787.7 444084.4 356343.3 489090.4 1230629 1643594 866418.2 Anthranilate 1872647 8827103 10300000 2512118 1754224 2810072 2743607 2339228 2317618 3074926 2670855 1349049 Picolinic acid 5697850 5468235 5132160 5651640 6059556 4786610 5196138 5919378 5397002 5070434 4947866 5474274 g-Oxalo-crotonate 17400000 12300000 23000000 23100000 16700000 24300000 31200000 23900000 22200000 20800000 25900000 11300000 Formyl-5-hydroxy-kynurenamine 4303696 1624258 2353268 5552108 4612482 5666338 6346780 5389814 6029606 10600000 13200000 7945914 Hydroxyindole-acetylglycine 29460.27 17027.29 27203.62 32826.7 46286.27 41925.05 51849.01 39880.16 28087.94 36204.3 42188.82 15222.45 Kynurenate 247151.8 331872.8 376158.8 217843.2 395637.8 313797.4 250834.6 279259.8 253730.4 265516.3 219812 282174.9 5-hydroxytryptophan 357986.8 408908.6 445912.7 408035.8 368746 436405.8 408396.4 395606.2 348659.9 362454.8 410447.4 202044 L-tryptophan 69214660 82505580 101126700 105818000 75851700 105405200 119800400 101846100 87619690 61683320 77534970 36570020 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID m/z RT 5-Hydroxyindoleacetate C05635 192.0618 0.5667723 3-methyldioxyindole C05834 164.0704 0.8904966 3-Methyleneoxindole C02796 146.0603 1.742382 Indole C00463 118.0656 1.748629 Indole-3-acetaldehyde C00637 160.0761 1.733175 Indole-3-acetate C00954 176.0708 1.934664 Indolepyruvate C00331 204.0636 0.877594 Indoxyl C05658 134.0603 0.8851032 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 Formyl-5-hydroxy-kynurenamine C05647 209.0922 1.325983 Hydroxyindole-acetylglycine C05832 249.085 1.591549 Kynurenate C01717 190.0499 0.9183065 5-hydroxytryptophan C00643 221.0923 1.048222 L-tryptophan C00078 205.0975 1.736209 METABOLITES_END #END