#METABOLOMICS WORKBENCH yli_20161229_171731 DATATRACK_ID:808 STUDY_ID:ST000528 ANALYSIS_ID:AN000806 PROJECT_ID:PR000388 VERSION 1 CREATED_ON December 30, 2016, 10:46 am #PROJECT PR:PROJECT_TITLE Regulation of Metabolism by LSR PR:PROJECT_TYPE LC-MS Lipidomics 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 University 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 9195416861 #STUDY ST:STUDY_TITLE Regulation of Metabolism by LSR ST:STUDY_TYPE LC-MS lipidomics 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 E. Cornwallis Road,Research Triangle Park, North Carolina, 27709, USA ST:EMAIL ssumner@rti.org ST:PHONE 919-541-7479 #SUBJECT SU:SUBJECT_TYPE Cells SU:SUBJECT_SPECIES Homo sapiens #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - POS_Lip_EQ_P28_JFtotPool1-6 Factor1 Cell Line:Study Pool DRCC ID=P28 SUBJECT_SAMPLE_FACTORS - POS_Lip_P_29_JFtotPool2 Factor1 Cell Line:Study Pool DRCC ID=P_29 SUBJECT_SAMPLE_FACTORS - POS_Lip_P_30_JFtotPool3 Factor1 Cell Line:Study Pool DRCC ID=P_30 SUBJECT_SAMPLE_FACTORS - POS_Lip_P_31_JFtotPool4 Factor1 Cell Line:Study Pool DRCC ID=P_31 SUBJECT_SAMPLE_FACTORS - POS_Lip_P_32_JFtotPool5 Factor1 Cell Line:Study Pool DRCC ID=P_32 SUBJECT_SAMPLE_FACTORS - POS_Lip_P_33_JFtotPool6 Factor1 Cell Line:Study Pool DRCC ID=P_33 SUBJECT_SAMPLE_FACTORS - POS_Lip_S_26_MCF7-CRISPR Factor1 Cell Line:MCF7 CRISPR DRCC ID=S_26 SUBJECT_SAMPLE_FACTORS - POS_Lip_S_27_MCF7-CRISPR Factor1 Cell Line:MCF7 CRISPR DRCC ID=S_27 SUBJECT_SAMPLE_FACTORS - POS_Lip_S_28_MCF7-CRISPR Factor1 Cell Line:MCF7 CRISPR DRCC ID=S_28 SUBJECT_SAMPLE_FACTORS - POS_Lip_S_29-2_MCF7-CRISPR Factor1 Cell Line:MCF7 CRISPR DRCC ID=S_29-2 SUBJECT_SAMPLE_FACTORS - POS_Lip_S_30_MCF7-CRISPR Factor1 Cell Line:MCF7 CRISPR DRCC ID=S_30 SUBJECT_SAMPLE_FACTORS - POS_Lip_S_21_MCF7-1 Factor1 Cell Line:MCF7 parental DRCC ID=S_21 SUBJECT_SAMPLE_FACTORS - POS_Lip_S_22_MCF7-2 Factor1 Cell Line:MCF7 parental DRCC ID=S_22 SUBJECT_SAMPLE_FACTORS - POS_Lip_S_23_MCF7 Factor1 Cell Line:MCF7 parental DRCC ID=S_23 SUBJECT_SAMPLE_FACTORS - POS_Lip_S_24_MCF7 Factor1 Cell Line:MCF7 parental DRCC ID=S_24 SUBJECT_SAMPLE_FACTORS - POS_Lip_S_25_MCF7 Factor1 Cell Line:MCF7 parental DRCC ID=S_25 SUBJECT_SAMPLE_FACTORS - POS_Lip_S_31_HS2 Factor1 Cell Line:Hs578t parental DRCC ID=S_31 SUBJECT_SAMPLE_FACTORS - POS_Lip_S_32_HS Factor1 Cell Line:Hs578t parental DRCC ID=S_32 SUBJECT_SAMPLE_FACTORS - POS_Lip_S_33_HS#2 Factor1 Cell Line:Hs578t parental DRCC ID=S_33 SUBJECT_SAMPLE_FACTORS - POS_Lip_S_34_HS#2 Factor1 Cell Line:Hs578t parental DRCC ID=S_34 SUBJECT_SAMPLE_FACTORS - POS_Lip_S_35_HS#2 Factor1 Cell Line:Hs578t parental DRCC ID=S_35 SUBJECT_SAMPLE_FACTORS - POS_Lip_S_36_LSRH-L2 Factor1 Cell Line:Hs578t+ DRCC ID=S_36 SUBJECT_SAMPLE_FACTORS - POS_Lip_S_37_LSRH-L4 Factor1 Cell Line:Hs578t+ DRCC ID=S_37 SUBJECT_SAMPLE_FACTORS - POS_Lip_S_38_LSRH-L6 Factor1 Cell Line:Hs578t+ DRCC ID=S_38 SUBJECT_SAMPLE_FACTORS - POS_Lip_S_39_LSRH-L8 Factor1 Cell Line:Hs578t+ DRCC ID=S_39 SUBJECT_SAMPLE_FACTORS - POS_Lip_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 The cells were cultured under obesogenic media conditions. #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 lipidomics, transferred the maximum clean volume of the lower SP:SAMPLEPREP_SUMMARY lipid-rich DCM layer to a new, pre-labeled LoBind tube and a 375 µL aliquot was SP:SAMPLEPREP_SUMMARY then transferred to a new 1.5 mL, LoBind tube for analysis. An additional 125 SP:SAMPLEPREP_SUMMARY µL aliquot of each study sample was transferred to a 7.0 mL glass vial to make SP:SAMPLEPREP_SUMMARY a QC pool, which was vortexed for 30 sec and aliquoted into 5 Total Pool samples SP:SAMPLEPREP_SUMMARY (375 µL each), and the remaining was used for 1 Equilibrium sample (column SP:SAMPLEPREP_SUMMARY conditioning). Samples were frozen at -80˚C for 60 mins and lyophilized to SP:SAMPLEPREP_SUMMARY dryness. Samples were reconstituted in ACN/IPA/H2O (65:30:5 v/v/v), vortexed at SP:SAMPLEPREP_SUMMARY 4,000 rpm for 2 mins and centrifuged at 16,000 rcf for 4 min. 225 µL of the SP:SAMPLEPREP_SUMMARY supernatant was transferred to pre-labeled autosampler vials and 10 µL was SP:SAMPLEPREP_SUMMARY injected into OrbiTrap Velos. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Lipidomics CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters Acquity CSH C18 (100 x 2.1mm, 1.7um) CH:FLOW_RATE 0.25 mL/min CH:COLUMN_TEMPERATURE 50 °C CH:SOLVENT_A 10mM Ammonium Formate in 60:40 H2O/ACN with 0.1% formic acid CH:SOLVENT_B 10mM Ammonium Formate in 90:10 IPA/ACN with 0.1% formic acid CH:COLUMN_PRESSURE 6000-7500psi CH:ANALYTICAL_TIME 30 min CH:WEAK_WASH_SOLVENT_NAME 60:40 water/ACN CH:STRONG_WASH_SOLVENT_NAME 90:10 IPA/ACN CH:TARGET_SAMPLE_TEMPERATURE 8 °C CH:SAMPLE_LOOP_SIZE 20 µL CH:RANDOMIZATION_ORDER Yes #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Thermo Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:CAPILLARY_TEMPERATURE 250 °C MS:COLLISION_ENERGY 35 eV MS:FRAGMENTATION_METHOD CID MS:IONIZATION ES+ MS:MASS_ACCURACY 10 ppm MS:SOURCE_TEMPERATURE 400 °C MS:SPRAY_VOLTAGE 4.0 kV MS:DATAFORMAT Profile MS:RESOLUTION_SETTING 30,000 MS:SCAN_RANGE_MOVERZ 120-2000 m/z MS:SCANNING_RANGE Normal #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS m/z MS_METABOLITE_DATA_START Samples POS_Lip_EQ_P28_JFtotPool1-6 POS_Lip_P_29_JFtotPool2 POS_Lip_P_30_JFtotPool3 POS_Lip_P_31_JFtotPool4 POS_Lip_P_32_JFtotPool5 POS_Lip_P_33_JFtotPool6 POS_Lip_S_26_MCF7-CRISPR POS_Lip_S_27_MCF7-CRISPR POS_Lip_S_28_MCF7-CRISPR POS_Lip_S_29-2_MCF7-CRISPR POS_Lip_S_30_MCF7-CRISPR POS_Lip_S_21_MCF7-1 POS_Lip_S_22_MCF7-2 POS_Lip_S_23_MCF7 POS_Lip_S_24_MCF7 POS_Lip_S_25_MCF7 POS_Lip_S_31_HS2 POS_Lip_S_32_HS POS_Lip_S_33_HS#2 POS_Lip_S_34_HS#2 POS_Lip_S_35_HS#2 POS_Lip_S_36_LSRH-L2 POS_Lip_S_37_LSRH-L4 POS_Lip_S_38_LSRH-L6 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9.54_383.3966m/z 22.09_831.5181m/z 9.50_425.4430m/z 12.47_1092.7848m/z 9.54_439.4583m/z 15.70_773.4713m/z 14.17_754.4010m/z 14.04_887.5520m/z 14.42_537.5055n 13.72_828.6405m/z 11.36_1115.2582m/z 1.17_247.1521m/z 13.46_698.3424m/z 13.48_187.5401m/z 14.62_708.3503n 13.97_746.2509m/z 12.20_690.5366m/z 16.18_1179.3090n 21.17_1468.4126m/z 6.02_582.4187m/z 12.39_795.8020m/z 13.46_737.7584m/z 19.43_652.2606m/z 14.32_275.6068m/z METABOLITES_END #END