#METABOLOMICS WORKBENCH ReemAlMalki91_20240702_085853 DATATRACK_ID:4969 STUDY_ID:ST003305 ANALYSIS_ID:AN005416 PROJECT_ID:PR002055 VERSION 1 CREATED_ON July 7, 2024, 10:47 pm #PROJECT PR:PROJECT_TITLE Comparative analysis of breast cancer metabolomes highlights fascin's central PR:PROJECT_TITLE role in regulating key pathways related to disease progression PR:PROJECT_SUMMARY Omics technologies provide useful tools for the identification of novel PR:PROJECT_SUMMARY biomarkers in many diseases, including breast cancer, which is the most PR:PROJECT_SUMMARY diagnosed cancer in women worldwide. We and others have reported a central role PR:PROJECT_SUMMARY for the actin-bundling protein (fascin) in regulating breast cancer disease PR:PROJECT_SUMMARY progression at different levels. However, whether fascin expression promotes PR:PROJECT_SUMMARY metabolic molecules that could predict disease progression has not been fully PR:PROJECT_SUMMARY elucidated. Here, fascin expression was manipulated via knockdown PR:PROJECT_SUMMARY (fascinKD+NORF) and rescue (fascinKD+FORF) in the naturally fascin-positive PR:PROJECT_SUMMARY (fascinpos+NORF) MDA-MB-231 breast cancer cells. Whether fascin dysregulates PR:PROJECT_SUMMARY metabolic profiles that are associated with disease progression was assessed PR:PROJECT_SUMMARY using untargeted metabolomics analyses via liquid chromatography-mass PR:PROJECT_SUMMARY spectrometry. An overall of 12,226 metabolites were detected in the tested cell PR:PROJECT_SUMMARY pellets. Fascinpos+NORF cell pellets showed 2,510 and 3,804 significantly PR:PROJECT_SUMMARY dysregulated metabolites compared to their fascinKD+NORF counterparts. Fascin PR:PROJECT_SUMMARY rescue (fascinKD+FORF) revealed 2,710 significantly dysregulated cellular PR:PROJECT_SUMMARY metabolites compared to fascinKD+NORF counterparts. 101 overlapped cellular PR:PROJECT_SUMMARY metabolites between fascinKD+FORF and fascinpos+NORF were significantly PR:PROJECT_SUMMARY dysregulated in the fascinKD+NORF cells. Analysis of the significantly PR:PROJECT_SUMMARY dysregulated metabolites by fascin expression revealed their involvement in the PR:PROJECT_SUMMARY metabolism of sphingolipid, phenylalanine, tyrosine and tryptophan biosynthesis, PR:PROJECT_SUMMARY and pantothenate and CoA biosynthesis, which are critical pathways for breast PR:PROJECT_SUMMARY cancer progression. Our findings of fascin-mediated alteration of metabolic PR:PROJECT_SUMMARY pathways could be used as putative poor prognostic biomarkers and highlight PR:PROJECT_SUMMARY other underlying mechanisms of fascin contribution to breast cancer progression. PR:INSTITUTE King Faisal Specialist Hospital and Research Centre (KFSHRC) PR:LAST_NAME AlMalki PR:FIRST_NAME Reem PR:ADDRESS Al Mather road, Riyadh, KSA, 11211, Saudi Arabia PR:EMAIL rgalmalki@kfshrc.edu.sa PR:PHONE +966534045397 #STUDY ST:STUDY_TITLE Comparative analysis of breast cancer metabolomes highlights fascin's central ST:STUDY_TITLE role in regulating key pathways related to disease progression ST:STUDY_SUMMARY Omics technologies provide useful tools for the identification of novel ST:STUDY_SUMMARY biomarkers in many diseases, including breast cancer, which is the most ST:STUDY_SUMMARY diagnosed cancer in women worldwide. We and others have reported a central role ST:STUDY_SUMMARY for the actin-bundling protein (fascin) in regulating breast cancer disease ST:STUDY_SUMMARY progression at different levels. However, whether fascin expression promotes ST:STUDY_SUMMARY metabolic molecules that could predict disease progression has not been fully ST:STUDY_SUMMARY elucidated. Here, fascin expression was manipulated via knockdown ST:STUDY_SUMMARY (fascinKD+NORF) and rescue (fascinKD+FORF) in the naturally fascin-positive ST:STUDY_SUMMARY (fascinpos+NORF) MDA-MB-231 breast cancer cells. Whether fascin dysregulates ST:STUDY_SUMMARY metabolic profiles that are associated with disease progression was assessed ST:STUDY_SUMMARY using untargeted metabolomics analyses via liquid chromatography-mass ST:STUDY_SUMMARY spectrometry. An overall of 12,226 metabolites were detected in the tested cell ST:STUDY_SUMMARY pellets. Fascinpos+NORF cell pellets showed 2,510 and 3,804 significantly ST:STUDY_SUMMARY dysregulated metabolites compared to their fascinKD+NORF counterparts. Fascin ST:STUDY_SUMMARY rescue (fascinKD+FORF) revealed 2,710 significantly dysregulated cellular ST:STUDY_SUMMARY metabolites compared to fascinKD+NORF counterparts. 101 overlapped cellular ST:STUDY_SUMMARY metabolites between fascinKD+FORF and fascinpos+NORF were significantly ST:STUDY_SUMMARY dysregulated in the fascinKD+NORF cells. Analysis of the significantly ST:STUDY_SUMMARY dysregulated metabolites by fascin expression revealed their involvement in the ST:STUDY_SUMMARY metabolism of sphingolipid, phenylalanine, tyrosine and tryptophan biosynthesis, ST:STUDY_SUMMARY and pantothenate and CoA biosynthesis, which are critical pathways for breast ST:STUDY_SUMMARY cancer progression. Our findings of fascin-mediated alteration of metabolic ST:STUDY_SUMMARY pathways could be used as putative poor prognostic biomarkers and highlight ST:STUDY_SUMMARY other underlying mechanisms of fascin contribution to breast cancer progression. ST:INSTITUTE King Faisal Specialist Hospital and Research Centre (KFSHRC) ST:LAST_NAME AlMalki ST:FIRST_NAME Reem ST:ADDRESS King Fahad road, Riyadh, KSA, 00000, Saudi Arabia ST:EMAIL rgalmalki@kfshrc.edu.sa ST:PHONE +966534045397 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #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 - CON_1_P Sample source:Breast cancer cells | Group:Control Sample type=Pellet; RAW_FILE_NAME(Raw data_NEG)=CON_1_P_NEG SUBJECT_SAMPLE_FACTORS - CON_2_P Sample source:Breast cancer cells | Group:Control Sample type=Pellet; RAW_FILE_NAME(Raw data_NEG)=CON_2_P_NEG SUBJECT_SAMPLE_FACTORS - CON_3_P Sample source:Breast cancer cells | Group:Control Sample type=Pellet; RAW_FILE_NAME(Raw data_NEG)=CON_3_P_NEG SUBJECT_SAMPLE_FACTORS - 40N_1_P Sample source:Breast cancer cells | Group:knockdown Sample type=Pellet; RAW_FILE_NAME(Raw data_NEG)=40N_1_P_NEG SUBJECT_SAMPLE_FACTORS - 40N_2_P Sample source:Breast cancer cells | Group:knockdown Sample type=Pellet; RAW_FILE_NAME(Raw data_NEG)=40N_2_P_NEG SUBJECT_SAMPLE_FACTORS - 40N_3_P Sample source:Breast cancer cells | Group:knockdown Sample type=Pellet; RAW_FILE_NAME(Raw data_NEG)=40N_3_P_NEG SUBJECT_SAMPLE_FACTORS - 40F_1_P Sample source:Breast cancer cells | Group:restore Sample type=Pellet; RAW_FILE_NAME(Raw data_NEG)=40F_1_P_NEG SUBJECT_SAMPLE_FACTORS - 40F_2_P Sample source:Breast cancer cells | Group:restore Sample type=Pellet; RAW_FILE_NAME(Raw data_NEG)=40F_2_P_NEG SUBJECT_SAMPLE_FACTORS - 40F_3_P Sample source:Breast cancer cells | Group:restore Sample type=Pellet; RAW_FILE_NAME(Raw data_NEG)=40F_3_P_NEG SUBJECT_SAMPLE_FACTORS - CON_1_S Sample source:Breast cancer cells | Group:Control Sample type=Secretome; RAW_FILE_NAME(Raw data_NEG)=CON_1_S_NEG SUBJECT_SAMPLE_FACTORS - CON_2_S Sample source:Breast cancer cells | Group:Control Sample type=Secretome; RAW_FILE_NAME(Raw data_NEG)=CON_2_S_NEG SUBJECT_SAMPLE_FACTORS - CON_3_S Sample source:Breast cancer cells | Group:Control Sample type=Secretome; RAW_FILE_NAME(Raw data_NEG)=CON_3_S_NEG SUBJECT_SAMPLE_FACTORS - 40F_1_S Sample source:Breast cancer cells | Group:restore Sample type=Secretome; RAW_FILE_NAME(Raw data_NEG)=40F_1_S_NEG SUBJECT_SAMPLE_FACTORS - 40F_2_S Sample source:Breast cancer cells | Group:restore Sample type=Secretome; RAW_FILE_NAME(Raw data_NEG)=40F_2_S_NEG SUBJECT_SAMPLE_FACTORS - 40F_3_S Sample source:Breast cancer cells | Group:restore Sample type=Secretome; RAW_FILE_NAME(Raw data_NEG)=40F_3_S_NEG SUBJECT_SAMPLE_FACTORS - 40N_1_S Sample source:Breast cancer cells | Group:knockdown Sample type=Secretome; RAW_FILE_NAME(Raw data_NEG)=40N_1_S_NEG SUBJECT_SAMPLE_FACTORS - 40N_2_S Sample source:Breast cancer cells | Group:knockdown Sample type=Secretome; RAW_FILE_NAME(Raw data_NEG)=40N_2_S_NEG SUBJECT_SAMPLE_FACTORS - 40N_3_S Sample source:Breast cancer cells | Group:knockdown Sample type=Secretome; RAW_FILE_NAME(Raw data_NEG)=40N_3_S_NEG SUBJECT_SAMPLE_FACTORS - QC_P_MDA_1 Sample source:Breast cancer cells | Group:QC Sample type=QC; RAW_FILE_NAME(Raw data_NEG)=QC_P_MDA_1_NEG SUBJECT_SAMPLE_FACTORS - QC_P_MDA_2 Sample source:Breast cancer cells | Group:QC Sample type=QC; RAW_FILE_NAME(Raw data_NEG)=QC_P_MDA_2_NEG SUBJECT_SAMPLE_FACTORS - QC_S_MDA_1 Sample source:Breast cancer cells | Group:QC Sample type=QC; RAW_FILE_NAME(Raw data_NEG)=QC_S_MDA_1_NEG SUBJECT_SAMPLE_FACTORS - QC_S_MDA_2 Sample source:Breast cancer cells | Group:QC Sample type=QC; RAW_FILE_NAME(Raw data_NEG)=QC_S_MDA_2_NEG #COLLECTION CO:COLLECTION_SUMMARY Cell pellet metabolites were extracted after removing the media and washing the CO:COLLECTION_SUMMARY cells with chilled 1x cold PBS as previously described [20]. The plates were CO:COLLECTION_SUMMARY dipped in liquid nitrogen to quench the metabolism and reduce the experimental CO:COLLECTION_SUMMARY variations. 1 mL of cold 80% methanol: water was added to each plate for CO:COLLECTION_SUMMARY metabolites extraction, and cells were detached using a cell scraper and CO:COLLECTION_SUMMARY transferred to 1.5 ml Eppendorf tubes. The mixtures were vortexed at 4°C, 600 CO:COLLECTION_SUMMARY rpm for 1h in Thermomixer (Eppendorf, Germany). The samples were spun down at CO:COLLECTION_SUMMARY 4°C, 16000 rpm for 10 min (Eppendorf, Germany). The supernatants (secretomes) CO:COLLECTION_SUMMARY were transferred to new Eppendorf tubes. Similarly, 900 µl of extraction CO:COLLECTION_SUMMARY solvent 1:1 (v/v) acetonitrile: methanol (ACN: MeOH) was added to 100 µl of CO:COLLECTION_SUMMARY media for secretome metabolites extraction. The mixtures were vortexed in CO:COLLECTION_SUMMARY Thermomixer (Eppendorf, Germany) at 600 rpm, 4ºC for 1 h. The samples were spun CO:COLLECTION_SUMMARY down at 16000 rpm, 4ºC for 10 min (Eppendorf, Germany), and then the secretomes CO:COLLECTION_SUMMARY were transferred to new Eppendorf tubes. The cell pellets and secretome extracts CO:COLLECTION_SUMMARY were completely evaporated in a SpeedVac (Christ, Germany) and stored at -80°C CO:COLLECTION_SUMMARY until LC-MS analysis [21,22] . CO:SAMPLE_TYPE Breast cancer cells #TREATMENT TR:TREATMENT_SUMMARY Gene knockdown and restoration MDA-MB-231 breast cancer cells are naturally TR:TREATMENT_SUMMARY fascin-positive. The establishment of stable fascin knockdown (fascinKD) and TR:TREATMENT_SUMMARY control (fascinpos) in MDA-MB-231 cells using fascin and scrambled shRNA, TR:TREATMENT_SUMMARY respectively, was previously described [doi:10.1371/journal.pone.0027339]. TR:TREATMENT_SUMMARY Furthermore, the rescue of fascin expression in the fascinKD MDA-MB-231 cells TR:TREATMENT_SUMMARY using fascin ORF (fascinKD+FORF) was also previously described TR:TREATMENT_SUMMARY [doi:10.1002/ijc.32183]. For transfection control, empty ORF was used in TR:TREATMENT_SUMMARY fascinKD (fascinKD+NORF) and fascinpos (fascinpos+NORF) cells. Fascin expression TR:TREATMENT_SUMMARY or knockdown was routinely checked at the RNA and protein levels. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Cell pellet metabolites were extracted after removing the media and washing the SP:SAMPLEPREP_SUMMARY cells with chilled 1x cold PBS as previously described SP:SAMPLEPREP_SUMMARY [doi:10.3390/ijms24044219]. The plates were dipped in liquid nitrogen to quench SP:SAMPLEPREP_SUMMARY the metabolism and reduce the experimental variations. 1 mL of cold 80% SP:SAMPLEPREP_SUMMARY methanol: water was added to each plate for metabolites extraction, and cells SP:SAMPLEPREP_SUMMARY were detached using a cell scraper and transferred to 1.5 ml Eppendorf tubes. SP:SAMPLEPREP_SUMMARY The mixtures were vortexed at 4°C, 600 rpm for 1h in Thermomixer (Eppendorf, SP:SAMPLEPREP_SUMMARY Germany). The samples were spun down at 4°C, 16000 rpm for 10 min (Eppendorf, SP:SAMPLEPREP_SUMMARY Germany). The supernatants (secretomes) were transferred to new Eppendorf tubes. SP:SAMPLEPREP_SUMMARY Similarly, 900 µl of extraction solvent 1:1 (v/v) acetonitrile: methanol (ACN: SP:SAMPLEPREP_SUMMARY MeOH) was added to 100 µl of media for secretome metabolites extraction. The SP:SAMPLEPREP_SUMMARY mixtures were vortexed in Thermomixer (Eppendorf, Germany) at 600 rpm, 4ºC for SP:SAMPLEPREP_SUMMARY 1 h. The samples were spun down at 16000 rpm, 4ºC for 10 min (Eppendorf, SP:SAMPLEPREP_SUMMARY Germany), and then the secretomes were transferred to new Eppendorf tubes. The SP:SAMPLEPREP_SUMMARY cell pellets and secretome extracts were completely evaporated in a SpeedVac SP:SAMPLEPREP_SUMMARY (Christ, Germany) and stored at -80°C until LC-MS analysis [21,22] . #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The dried extract samples were reconstituted in a 1:1 mobile phase (A: 0.1% CH:CHROMATOGRAPHY_SUMMARY formic acid in dH2O and B: 0.1% formic acid in (1:1) (v/v) MeOH: ACN) for an CH:CHROMATOGRAPHY_SUMMARY LC-MS metabolomics analysis [20]. First, 5 µL of the sample was introduced to CH:CHROMATOGRAPHY_SUMMARY the inlet technique, where the metabolites were separated in reversed-phase CH:CHROMATOGRAPHY_SUMMARY liquid chromatography using an ACQUITY UPLC XSelect (100 × 2.1 mm × 2.5 μm) CH:CHROMATOGRAPHY_SUMMARY column (Waters Ltd., Elstree, UK). The mobile phase flow rate was set at 300 CH:CHROMATOGRAPHY_SUMMARY μL/min, the column temperature maintained at 55 °C and the samples were CH:CHROMATOGRAPHY_SUMMARY maintained at 4 °C in the autosampler. Mobile phases A and B were pumped to the CH:CHROMATOGRAPHY_SUMMARY column in a gradient mode (0–16 min 95–5% A, 16–19 min 5% A, 19–20 min CH:CHROMATOGRAPHY_SUMMARY 5–95% A, and 20–22 min 5–95% A). The molecules eluted from the LC were CH:CHROMATOGRAPHY_SUMMARY positively or negatively ionized using an electrospray ionization source (ESI) CH:CHROMATOGRAPHY_SUMMARY and separated in the gas phase based on m/z using a Xevo G2-S QTOF mass CH:CHROMATOGRAPHY_SUMMARY spectrometer (Waters Ltd., Elstree, UK). The metabolites were ionized in the ESI CH:CHROMATOGRAPHY_SUMMARY source, where the source temperature was 150 °C, the desolvation temperature CH:CHROMATOGRAPHY_SUMMARY was 500 °C, and the capillary voltages were kept at 3.20 kV (ESI+) or 3 kV CH:CHROMATOGRAPHY_SUMMARY (ESI−). The cone voltage was 40 V. the desolvation gas flow was 800.0 L/h, and CH:CHROMATOGRAPHY_SUMMARY the cone gas flow was 50 L/h. The collision energies of the low and high CH:CHROMATOGRAPHY_SUMMARY functions were set to off and 10–50 V, respectively, in the MSE CH:CHROMATOGRAPHY_SUMMARY data-independent acquisition (DIA) mode. The mass spectrometer was calibrated, CH:CHROMATOGRAPHY_SUMMARY as recommended by the vendor, with sodium formate in the range of 100–1200 Da CH:CHROMATOGRAPHY_SUMMARY in both ionization modes. The lock mass compound, leucine-enkephaline (an CH:CHROMATOGRAPHY_SUMMARY external reference to the ion m/z 556.2771 in (ESI+) and 554.2615 (ESI−)), was CH:CHROMATOGRAPHY_SUMMARY injected continuously, switching between the sample and the reference every 45 CH:CHROMATOGRAPHY_SUMMARY and 60 s for ESI+ and ESI−, respectively, for a 0.5 s scan time, a flow rate CH:CHROMATOGRAPHY_SUMMARY of 10 µL/min, a cone voltage of 30 V, and a collision energy of 4 V. DIA were CH:CHROMATOGRAPHY_SUMMARY collected in continuum mode with a Masslynx™ V4.1 workstation (Waters Inc., CH:CHROMATOGRAPHY_SUMMARY Milford, MA, USA). Quality control samples (QCs) were performed by pooling 10 CH:CHROMATOGRAPHY_SUMMARY µL from each study sample and extracted, after that, introduced to the CH:CHROMATOGRAPHY_SUMMARY instrument with randomization to validate the system's stability. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters XSelect HSS C18 (100 × 2.1mm, 2.5um) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 50% methanol:50% acetonitrile; 0.1% formic acid CH:FLOW_GRADIENT 0–16 min 95–5% A, 16–19 min 5% A, 19–20 min 5–95% A, and 20–22 min CH:FLOW_GRADIENT 5–95% A CH:FLOW_RATE 0.300 mL/min CH:COLUMN_TEMPERATURE 55 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Xevo-G2-XS MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS The DIA data were collected with a Masslynx™ V4.1 workstation in continuum MS:MS_COMMENTS mode (Waters Inc., Milford, MA, USA). The raw MS data were processed following a MS:MS_COMMENTS standard pipeline using the Progenesis QI v.3.0 software. MS:MS_RESULTS_FILE ST003305_AN005416_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END