#METABOLOMICS WORKBENCH yli_20161230_071013 DATATRACK_ID:809 STUDY_ID:ST000529 ANALYSIS_ID:AN000807 PROJECT_ID:PR000388 VERSION 1 CREATED_ON December 30, 2016, 8:07 am #PROJECT PR:PROJECT_TITLE Regulation of Metabolism by LSR PR:PROJECT_TYPE Broad spectrum, reverse phase LCMS metabolomics PR:PROJECT_SUMMARY Breast cancer is a global crisis, accounting for nearly one quarter of all PR:PROJECT_SUMMARY cancers in women. In the U.S., African-American (AA) women suffer PR:PROJECT_SUMMARY disproportionately from breast cancer mortality compared to other racial/ethnic PR:PROJECT_SUMMARY groups. Both social (socioeconomic factors, diet, obesity) and biological PR:PROJECT_SUMMARY hypotheses (gene polymorphisms, gene expression profiling) have been advanced to PR:PROJECT_SUMMARY explain these discrepancies, but the differences remain poorly understood. PR:PROJECT_SUMMARY Multiple aspects of tumor aggressiveness have been identified in the AA PR:PROJECT_SUMMARY population, including a high proportion of basal-like tumors. Basal-like tumors PR:PROJECT_SUMMARY are clinically the most aggressive, characterized by enhanced cancer stem PR:PROJECT_SUMMARY cell-like features. Currently, no effective molecular therapies exist for these PR:PROJECT_SUMMARY highly aggressive cancers and patient survival is poor. Escalating this PR:PROJECT_SUMMARY disparity is the disease promoting effects of obesity and metabolic syndrome, PR:PROJECT_SUMMARY which are significantly higher in AA women. Obesity and its associated PR:PROJECT_SUMMARY inflammation have been attributed to poor patient outcomes, resistance to PR:PROJECT_SUMMARY chemotherapeutics, and/or cancer risk. A meta-analysis of 43 studies of obesity PR:PROJECT_SUMMARY and breast cancer revealed that obese patients were 33% more likely than PR:PROJECT_SUMMARY non-obese patients to die of breast cancer. Yet apart from correlative studies, PR:PROJECT_SUMMARY no reports have combined these factors with cancer disparities to ascertain PR:PROJECT_SUMMARY their molecular interactions and physiology on breast tumorigenesis. We tested PR:PROJECT_SUMMARY the integration these factors, with focus on a specific molecule, Lipolysis PR:PROJECT_SUMMARY Stimulated Lipoprotein Receptor (LSR), in the promotion of aggressive cancer PR:PROJECT_SUMMARY behaviors. LSR is a cell surface molecule that regulates post-prandial lipid PR:PROJECT_SUMMARY uptake in the liver, is sensitive to high fat diets, and is regulated by PR:PROJECT_SUMMARY metabolic cues, including leptin. By testing each of these factors as well as PR:PROJECT_SUMMARY their dynamic interactions on breast cancer tumorigenesis, we will gain valuable PR:PROJECT_SUMMARY insight into biological mechanisms that influence cancer risk/disparities, PR:PROJECT_SUMMARY response to therapy, and ultimately patient outcome. We recently reported LSR is PR:PROJECT_SUMMARY overexpressed in breast tumors, directs aggressive breast cancer cell behaviors PR:PROJECT_SUMMARY including proliferation and migration, and enhances cancer stem cell-like and PR:PROJECT_SUMMARY chemotherapeutic resistance features in breast cancer cells. PR:INSTITUTE North Carolina Central Univeristy PR:DEPARTMENT Department of Biology PR:LAST_NAME Fleming PR:FIRST_NAME Jodie PR:ADDRESS 1801 Fayetteville Street, Durham, NC 27707 PR:EMAIL Jodie.Fleming@nccu.edu PR:PHONE 919-530-6216 #STUDY ST:STUDY_TITLE Regulation of Metabolism by LSR ST:STUDY_TYPE Broad spectrum, reverse phase LCMS metabolomics (Negative ion mode) ST:STUDY_SUMMARY Our aim is to identify the LSR-driven metabolomics profile of breast cancer ST:STUDY_SUMMARY cells in lean and obesogenic environments. Breast cancer cell models with high ST:STUDY_SUMMARY or undetectable levels of LSR, including drug resistance models, were cultured ST:STUDY_SUMMARY in lean and obesogenic environments and comprehensive metabolomics profiling, ST:STUDY_SUMMARY including lipidomics-focused sub-analyses were performed. The metabolomics ST:STUDY_SUMMARY analyses using both approaches will help us determine if LSR enhances aggressive ST:STUDY_SUMMARY breast cancer phenotypes via modulation of cellular bioenergetic metabolism, ST:STUDY_SUMMARY ultimately contributing to poor patient outcome. ST:INSTITUTE RTI International ST:DEPARTMENT Systems and Translational Sciences ST:LABORATORY NIH Eastern Regional Comphrehensive Metabolomics Resource Core ST:LAST_NAME Sumner ST:FIRST_NAME Susan ST:ADDRESS 3040 East Cornwallis Road, RTI International, Research Triangle Park, North ST:ADDRESS Carolina, 27709, USA ST:EMAIL ssumner@rti.org ST:PHONE 919-541-7479 ST:NUM_GROUPS 4 ST:TOTAL_SUBJECTS 20 #SUBJECT SU:SUBJECT_TYPE Cells SU:SUBJECT_SPECIES Homo sapiens SU:SUBJECT_COMMENTS Cell pellets stored at -80˚C. SU:CELL_COUNTS 1E 7 cells/pellet #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - NEG_RP_EQ _P28_JFtotPool1-6 Factor1 Cell Line:Study Pool DRCC ID=P_28 SUBJECT_SAMPLE_FACTORS - NEG_RP_P_29_JFtotPool2 Factor1 Cell Line:Study Pool DRCC ID=P_29 SUBJECT_SAMPLE_FACTORS - NEG_RP_P_30_JFtotPool3 Factor1 Cell Line:Study Pool DRCC ID=P_30 SUBJECT_SAMPLE_FACTORS - NEG_RP_P_31_JFtotPool4 Factor1 Cell Line:Study Pool DRCC ID=P_31 SUBJECT_SAMPLE_FACTORS - NEG_RP_S_28_MCF7-CRISPR Factor1 Cell Line:MCF7 CRISPR DRCC ID=S_28 SUBJECT_SAMPLE_FACTORS - NEG_RP_S_30_MCF7-CRISPR Factor1 Cell Line:MCF7 CRISPR DRCC ID=S_30 SUBJECT_SAMPLE_FACTORS - NEG_RP_S_21_MCF7-1 Factor1 Cell Line:MCF7 parental DRCC ID=S_21 SUBJECT_SAMPLE_FACTORS - NEG_RP_S_22_MCF7-2 Factor1 Cell Line:MCF7 parental DRCC ID=S_22 SUBJECT_SAMPLE_FACTORS - NEG_RP_S_25_MCF7 Factor1 Cell Line:MCF7 parental DRCC ID=S_25 SUBJECT_SAMPLE_FACTORS - NEG_RP_S_31_HS2 Factor1 Cell Line:Hs578t parental DRCC ID=S_31 SUBJECT_SAMPLE_FACTORS - NEG_RP_S_32_HS Factor1 Cell Line:Hs578t parental DRCC ID=S_32 SUBJECT_SAMPLE_FACTORS - NEG_RP_S_33_HS#2 Factor1 Cell Line:Hs578t parental DRCC ID=S_33 SUBJECT_SAMPLE_FACTORS - NEG_RP_S_34_HS#2 Factor1 Cell Line:Hs578t parental DRCC ID=S_34 SUBJECT_SAMPLE_FACTORS - NEG_RP_S_35_HS#2 Factor1 Cell Line:Hs578t parental DRCC ID=S_35 SUBJECT_SAMPLE_FACTORS - NEG_RP_S_36_LSRH-L2 Factor1 Cell Line:Hs578t+ DRCC ID=S_36 SUBJECT_SAMPLE_FACTORS - NEG_RP_S_38_LSRH-L6 Factor1 Cell Line:Hs578t+ DRCC ID=S_38 SUBJECT_SAMPLE_FACTORS - NEG_RP_S_39_LSRH-L8 Factor1 Cell Line:Hs578t+ DRCC ID=S_39 SUBJECT_SAMPLE_FACTORS - NEG_RP_S_40_LSRH-L10 Factor1 Cell Line:Hs578t+ DRCC ID=S_40 #COLLECTION CO:COLLECTION_SUMMARY 20 breast cancer cell pellets from four cell lines [MCF7 parental (LSR+), MCF7 CO:COLLECTION_SUMMARY Crisper (LSR-), Hs578t parental (LSR-) and Hs578t (LSR++)], with five replicate CO:COLLECTION_SUMMARY samples for each cell line were stored at -80˚ until sample preparation. CO:SAMPLE_TYPE Cell pellets CO:STORAGE_CONDITIONS -80˚C #TREATMENT TR:TREATMENT_SUMMARY Cultured in an obesogenic media. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Cell pellets were resuspended in volume of ice cold Lipid Extractions Solvent SP:SAMPLEPREP_SUMMARY (50 µg/mL) based on biomass. Contents were transferred to new, pre-labeled 2.0 SP:SAMPLEPREP_SUMMARY mL LoBind tube with 10-15 ceramic beads, and homogenized using 2 pulses at 2,000 SP:SAMPLEPREP_SUMMARY rpm for 30 sec on a MagNA Lyser. Volume of HPLC-grade water (with 0.02 mg/mL SP:SAMPLEPREP_SUMMARY Tryptophan-d5 in water) was added based on biomass. Samples were allowed to sit SP:SAMPLEPREP_SUMMARY at room temperature for 10 minutes, then centrifuged at 16,000 rcf for 10 min at SP:SAMPLEPREP_SUMMARY 10˚C. For reverse phase, transferred the maximum clean volume of the upper SP:SAMPLEPREP_SUMMARY aqueous layer to a new, pre-labeled LoBind tube then a 150 µL aliquot was SP:SAMPLEPREP_SUMMARY transferred to a new 1.5 mL, LoBind tube for analysis. An additional 50 µL SP:SAMPLEPREP_SUMMARY aliquot of each study sample was combined in a 2 mL LoBind tube to make a QC SP:SAMPLEPREP_SUMMARY pool, which was vortexed for 30 sec and aliquoted into 5 Total Pool samples (150 SP:SAMPLEPREP_SUMMARY µL each), and the remainder used for 1 Equilibrium sample (for column SP:SAMPLEPREP_SUMMARY conditioning). Samples were placed at -80˚C for 60 mins and lyophilized to SP:SAMPLEPREP_SUMMARY dryness. Samples were reconstituted in H2O/Methanol (95:5 v/v), vortexed at SP:SAMPLEPREP_SUMMARY 5,000 rpm for 2 mins and centrifuged at 16,000 rcf for 4 min. 100 µL of each SP:SAMPLEPREP_SUMMARY supernatant was transferred to pre-labeled autosampler vials and 5 µL was SP:SAMPLEPREP_SUMMARY injected Synapt G2Si ESI-Q-TOF for data acquisition. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Reversed-Phase Gradient Seperation CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters Acquity HSS T3 (100 x 2.1mm, 1.8um) CH:FLOW_GRADIENT " Time(min) Flow Rate(mL/min) %A %B Curve 1. Initial 0.400 99.0 1.0 2. 1.00 CH:FLOW_GRADIENT 0.400 99.0 1.0 3. 16.00 0.400 1.0 99.0 4. 20.00 0.400 1.0 99.0 5. 20.50 0.400 CH:FLOW_GRADIENT 99.0 1.0 6. 22.00 0.400 99.0 1.0" CH:FLOW_RATE 0.4 mL/min CH:COLUMN_TEMPERATURE 50 °C CH:METHODS_FILENAME RTI-RCMRC-RP CH:SOLVENT_A Water with 0.1% formic acid CH:SOLVENT_B Methanol with 0.1% formic acid CH:METHODS_ID RTI-RCMRC-RP CH:INTERNAL_STANDARD L-Tryptophan-d5 CH:ANALYTICAL_TIME 22 min CH:WEAK_WASH_SOLVENT_NAME 10% MeOH CH:WEAK_WASH_VOLUME 1000 µL CH:STRONG_WASH_SOLVENT_NAME 80% MeOH CH:STRONG_WASH_VOLUME 1000 µL CH:TARGET_SAMPLE_TEMPERATURE 8 °C CH:SAMPLE_LOOP_SIZE 10 µL CH:SAMPLE_SYRINGE_SIZE 100 µL CH:RANDOMIZATION_ORDER Yes #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Waters Synapt G2 Si QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:CAPILLARY_TEMPERATURE 110 °C MS:CAPILLARY_VOLTAGE 1.0 kV MS:COLLISION_ENERGY 4 MS:FRAGMENTATION_METHOD CID MS:HELIUM_FLOW 180 MS:IONIZATION ES- MS:IONIZATION_POTENTIAL 5 ppm MS:SOURCE_TEMPERATURE 100 °C MS:DATAFORMAT Continuum MS:DESOLVATION_GAS_FLOW 400 L/Hr MS:DESOLVATION_TEMPERATURE 400 °C MS:RESOLUTION_SETTING 18000 MS:SCAN_RANGE_MOVERZ 50-1000 m/z MS:SCANNING_CYCLE 1.0 s MS:TUBE_LENS_VOLTAGE 74 #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS m/z MS_METABOLITE_DATA_START Samples NEG_RP_EQ _P28_JFtotPool1-6 NEG_RP_P_29_JFtotPool2 NEG_RP_P_30_JFtotPool3 NEG_RP_P_31_JFtotPool4 NEG_RP_S_28_MCF7-CRISPR NEG_RP_S_30_MCF7-CRISPR NEG_RP_S_21_MCF7-1 NEG_RP_S_22_MCF7-2 NEG_RP_S_25_MCF7 NEG_RP_S_31_HS2 NEG_RP_S_32_HS NEG_RP_S_33_HS#2 NEG_RP_S_34_HS#2 NEG_RP_S_35_HS#2 NEG_RP_S_36_LSRH-L2 NEG_RP_S_38_LSRH-L6 NEG_RP_S_39_LSRH-L8 NEG_RP_S_40_LSRH-L10 Factors Factor1 Cell Line:Study Pool Factor1 Cell Line:Study Pool Factor1 Cell Line:Study Pool Factor1 Cell Line:Study Pool Factor1 Cell Line:MCF7 CRISPR Factor1 Cell Line:MCF7 CRISPR Factor1 Cell Line:MCF7 parental Factor1 Cell Line:MCF7 parental Factor1 Cell Line:MCF7 parental Factor1 Cell Line:Hs578t parental Factor1 Cell Line:Hs578t parental Factor1 Cell Line:Hs578t parental Factor1 Cell Line:Hs578t parental Factor1 Cell Line:Hs578t parental Factor1 Cell Line:Hs578t+ Factor1 Cell Line:Hs578t+ Factor1 Cell Line:Hs578t+ Factor1 Cell Line:Hs578t+ 0.89_611.0405m/z 112.9244369 119.9782323 189.2982201 162.6998105 53.57154317 51.3438768 255.0026661 300.4791315 350.4437081 43.78838014 90.19664083 59.31283278 79.96138706 45.41792717 98.19685396 95.51229395 139.2569357 82.98115874 13.34_284.9382m/z 44.09096546 82.27762115 67.53067773 75.14943868 74.88774905 107.4131464 46.16847511 64.24385749 62.68812143 91.035868 131.147241 70.88010403 75.11315136 121.8211802 79.37272217 74.3692928 98.28563076 51.49991475 15.99_733.8628n 411.6429321 358.4942153 353.7903443 214.5832297 332.405328 277.4854147 340.8892482 335.3627466 166.7628183 418.802765 348.7476213 298.7421577 352.1019334 336.4486632 374.3698592 441.3694678 482.3037468 416.8415641 0.78_778.4604m/z 92.54425643 82.93311005 50.55290869 66.59161995 153.9760088 223.3909236 0 0 0 168.0764395 245.1051169 148.3279295 176.5130485 84.02128623 80.37596033 101.4520819 78.82122614 85.59284958 0.73_709.1720m/z 22.49447767 12.02836199 17.41964394 16.06340473 0 0.006520569 120.0229376 126.2837115 0.970129865 29.10399819 16.11908609 0 0.018134467 2.320295475 0 0 0 0 4.10_435.2158m/z 127.5669415 83.24090048 90.72651157 72.82382493 100.0829534 100.8332683 101.5123971 109.0032036 106.9455329 207.1863145 115.4933851 105.5243086 78.35405956 57.41670633 74.66343957 111.0186234 90.76327909 80.46912626 1.59_941.5365m/z 165.1428715 117.5140491 94.89450518 106.6816 216.9427142 215.5056923 0 12.81617342 90.1007186 162.5444458 182.1973951 89.09417245 91.29204007 117.9320631 166.8625822 170.0885483 173.3716301 163.9089324 2.68_460.1137m/z 53.06438165 28.75622594 39.41843161 37.29015751 0 0 140.3607402 94.82771567 0 119.2601548 40.32045244 47.5977269 51.19465705 34.45045926 0 0 0 0 1.25_415.9692m/z 63.16369965 87.15377357 107.4687476 114.0867418 5.34924496 7.571191786 71.73895551 102.5988229 178.2897604 72.18351615 172.7455408 52.04736003 45.37618525 181.6460256 19.57198133 22.43310774 74.64865639 30.36880087 1.10_436.9942m/z 21.03191136 30.04516309 17.01588532 21.02766984 36.80951408 41.96093834 7.299301834 29.24074096 31.72464582 46.70497809 32.73608274 17.93523937 44.34309022 86.64395773 12.50384296 0 0 0 2.49_634.0695m/z 185.7582413 124.5149379 140.3503045 102.9863198 0 0 12.89217802 20.06034183 0 372.403913 481.8253648 70.4234955 67.69032622 24.57491781 66.73802581 358.1141526 368.3286377 183.6679612 2.82_495.0059m/z 21.90248599 19.19030189 23.21509106 12.46305634 0 0 0 0 0 0 0 0 0 0 129.9583065 155.8059855 139.8733132 105.8995664 3.43_683.1658m/z 256.5617176 182.660702 162.6705026 149.86579 56.46256888 62.20859407 254.0635972 231.3763707 85.74827114 91.33836182 97.44260286 109.830496 90.26495679 136.4970071 328.9412073 662.3812865 739.8425056 620.5949424 0.67_497.0449m/z 186.4998275 183.273591 139.4490421 105.8570372 91.65241049 84.85591749 119.702136 143.8144133 170.6373172 93.37799849 32.80795987 81.9418761 57.68016095 153.7703188 149.7911548 215.1435927 259.9667117 195.1397098 6.80_964.9410m/z 116.8892561 114.5236169 89.81243236 65.41306737 0 0 0 0 0 754.2589207 1076.13546 534.4429251 449.8785814 280.6767513 0 0 0 0 1.30_818.0413m/z 261.3275078 295.917719 263.7177002 156.8443805 122.2900715 135.3801946 285.0268954 315.2702405 183.4338541 239.0645444 197.3414871 58.08503592 40.08506407 72.91044654 291.1663911 374.9412495 400.4040386 288.4297031 15.67_501.2988m/z 158.6570983 88.81552588 124.6401608 104.7875597 326.8600926 224.7782163 6.612100028 12.14046127 212.4614906 103.1556118 197.4475551 317.8708846 281.8484472 363.2629624 58.5293292 43.80109537 115.3390241 17.3712411 10.21_173.0246m/z 31.24049662 18.3229025 28.50459135 34.63667389 12.34614288 26.13852156 22.43322993 18.06725703 11.66132332 31.79390903 44.53318387 37.28899852 34.36310041 44.10512408 28.24865901 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