#METABOLOMICS WORKBENCH ahmet_mmtv_20230626_161648 DATATRACK_ID:4121 STUDY_ID:ST002748 ANALYSIS_ID:AN004458 PROJECT_ID:PR001711 VERSION 1 CREATED_ON June 26, 2023, 6:05 pm #PROJECT PR:PROJECT_TITLE HER2 related MMTV-Neu mammary ducts studies PR:PROJECT_TYPE MS quantitative analysis PR:PROJECT_SUMMARY Metabolomics analysis of intact mammary ducts. HER2 is a driver oncogene PR:PROJECT_SUMMARY overexpressed in the majority of premalignant breast tumors known as ductal PR:PROJECT_SUMMARY carcinoma in situ (DCIS). Due to their stemness features, breast cancer stem PR:PROJECT_SUMMARY cells (BCSC) are considered the main drivers of breast tumor initiation and PR:PROJECT_SUMMARY progression. Here, we used clinical samples and mouse models of HER2+ breast PR:PROJECT_SUMMARY tumorigenesis to demonstrate that neither BCSCs nor their cell-of-origin express PR:PROJECT_SUMMARY HER2/Neu in early-stage breast tumors. Instead, our results demonstrate that Neu PR:PROJECT_SUMMARY overexpression results in the transformation of BCSCs in a non-cell autonomous PR:PROJECT_SUMMARY manner via triggering DNA damage and somatic mutagenesis in their Neu-negative PR:PROJECT_SUMMARY cell-of-origin. This is caused by the increased oxidative stress in the tissue PR:PROJECT_SUMMARY microenvironment generated by altered energy metabolism and increased reactive PR:PROJECT_SUMMARY oxygen species level in Neu-overexpressing mammary ducts. Therefore, our PR:PROJECT_SUMMARY findings illustrate a previously unrecognized mechanism of HER2-induced breast PR:PROJECT_SUMMARY tumor initiation, which may have an impact on future preventive treatments for PR:PROJECT_SUMMARY patients with HER2+ DCIS. PR:INSTITUTE The University of Manchester PR:DEPARTMENT Faculty of Biology, Medicine and Health PR:LABORATORY Ahmet Ucar Lab PR:LAST_NAME Ou PR:FIRST_NAME Yaqing PR:ADDRESS Oxford Road, Manchester, Great Manchester, M13 9PL, United Kingdom PR:EMAIL yaqing.ou@manchester.ac.uk PR:PHONE 07579759914 PR:CONTRIBUTORS Sevim B. Gurler, Oliver Wagstaff, Lili Dimitrova, Fuhui Chen, Robert Pedley, PR:CONTRIBUTORS William Weston, Ian Donaldson, Brian A. Telfer, David Novo, Kyriaki Pavlou, PR:CONTRIBUTORS George Taylor, Yaqing Ou, Kaye J. Williams, Andrew Gilmore, Keith Brennan, Ahmet PR:CONTRIBUTORS Ucar #STUDY ST:STUDY_TITLE HER2 overexpression initiates breast tumorigenesis non-cell autonomously by ST:STUDY_TITLE inducing oxidative stress in the tissue microenvironment ST:STUDY_SUMMARY HER2 is a driver oncogene overexpressed in the majority of premalignant breast ST:STUDY_SUMMARY tumors known as ductal carcinoma in situ (DCIS). Due to their stemness features, ST:STUDY_SUMMARY breast cancer stem cells (BCSC) are considered the main drivers of breast tumor ST:STUDY_SUMMARY initiation and progression. Here, we used clinical samples and mouse models of ST:STUDY_SUMMARY HER2+ breast tumorigenesis to demonstrate that neither BCSCs nor their ST:STUDY_SUMMARY cell-of-origin express HER2/Neu in early-stage breast tumors. Instead, our ST:STUDY_SUMMARY results demonstrate that Neu overexpression results in the transformation of ST:STUDY_SUMMARY BCSCs in a non-cell autonomous manner via triggering DNA damage and somatic ST:STUDY_SUMMARY mutagenesis in their Neu-negative cell-of-origin. This is caused by the ST:STUDY_SUMMARY increased oxidative stress in the tissue microenvironment generated by altered ST:STUDY_SUMMARY energy metabolism and increased reactive oxygen species level in ST:STUDY_SUMMARY Neu-overexpressing mammary ducts. Therefore, our findings illustrate a ST:STUDY_SUMMARY previously unrecognized mechanism of HER2-induced breast tumor initiation, which ST:STUDY_SUMMARY may have an impact on future preventive treatments for patients with HER2+ DCIS. ST:INSTITUTE The University of Manchester ST:DEPARTMENT Manchester Breast Centre ST:LABORATORY Ahmet Ucar Lab ST:LAST_NAME Ucar ST:FIRST_NAME Ahmet ST:ADDRESS Oxford Road, Manchester, M13 9PL, UK. ST:EMAIL ahmet.ucar@manchester.ac.uk ST:PHONE +44 (0)161 3067116 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:AGE_OR_AGE_RANGE 8-weeks old #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 - 407 660 Genotype:Wild-type | Treatment:Control RAW_FILE_NAME=407 660.wiff SUBJECT_SAMPLE_FACTORS - 407 661 Genotype:Wild-type | Treatment:Control RAW_FILE_NAME=407 661.wiff SUBJECT_SAMPLE_FACTORS - 407 662 Genotype:Mutant | Treatment:MMTV-NIC RAW_FILE_NAME=407 662.wiff SUBJECT_SAMPLE_FACTORS - 407 663 Genotype:Mutant | Treatment:MMTV-NIC RAW_FILE_NAME=407 663.wiff SUBJECT_SAMPLE_FACTORS - 407 667 Genotype:Mutant | Treatment:MMTV-NIC RAW_FILE_NAME=407 667.wiff SUBJECT_SAMPLE_FACTORS - 407 668 Genotype:Mutant | Treatment:MMTV-NIC RAW_FILE_NAME=407 668.wiff SUBJECT_SAMPLE_FACTORS - 407 669 Genotype:Wild-type | Treatment:Control RAW_FILE_NAME=407 669.wiff #COLLECTION CO:COLLECTION_SUMMARY No:4 mammary glands were dissected and spread on sterile microscope slides. CO:COLLECTION_SUMMARY Lymph nodes were removed, and the glands were cut into thin horizontal stripes CO:COLLECTION_SUMMARY before digesting them with Collagenase A (#11088793001, Roche) at 37°C for 3-4 CO:COLLECTION_SUMMARY hours. The ductal organoids were then separated from single cells using a 100-um CO:COLLECTION_SUMMARY mesh and collected into a tube by flushing them with medium. CO:SAMPLE_TYPE Epithelial cells #TREATMENT TR:TREATMENT_SUMMARY The mouse model of HER2+ breast cancer, MMTV-Neu-IRES-Cre (MMTV-NIC), is used as TR:TREATMENT_SUMMARY the treatment group. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Ductal organoids were centrifuged, tissue pellets were snap-frozen in liquid SP:SAMPLEPREP_SUMMARY nitrogen and then resuspended in 50% Methanol to incubate overnight at 4 °C. SP:SAMPLEPREP_SUMMARY Next day, samples were centrifuged at 20,000 x g for 3 min and the top 50 µl SP:SAMPLEPREP_SUMMARY supernatant was transferred to a glass autosampler vial with 300 µl insert and SP:SAMPLEPREP_SUMMARY capped. Quality control samples were made by pooling 10 µl from each sample. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Liquid chromatography-mass spectrometry analysis was performed using a CH:CHROMATOGRAPHY_SUMMARY Thermo-Fisher Ultimate 3000 HPLC system consisting of an HPG-3400RS high CH:CHROMATOGRAPHY_SUMMARY pressure gradient pump, TCC 3000SD column compartment and WPS 3000 Autosampler, CH:CHROMATOGRAPHY_SUMMARY coupled to a SCIEX 6600 TripleTOF Q-TOF mass spectrometer with TurboV ion CH:CHROMATOGRAPHY_SUMMARY source. The system was controlled by SCIEX Analyst 1.7.1, DCMS Link and CH:CHROMATOGRAPHY_SUMMARY Chromeleon Xpress software. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo-Fisher Ultimate 3000 HPLC CH:COLUMN_NAME Agilent Poroshell 120 HILIC-Z CH:SOLVENT_A 100% water; 10 mM ammonium acetate adjusted to pH 9 with ammonium hydroxide and CH:SOLVENT_A 20 µM medronic acid CH:SOLVENT_B 85% acetonitrile/15% water; 10 mM ammonium acetate adjusted to pH 9 with CH:SOLVENT_B ammonium hydroxide and 20 µM medronic acid CH:FLOW_GRADIENT flow rate of 0.25 ml/min, starting at 96 % B for 2 minutes, ramping to 65 % B CH:FLOW_GRADIENT over 20 min, hold at 65 % B for 2 min, then back to 96 % B CH:FLOW_RATE 0.25 ml/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6600 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS - MS:MS_RESULTS_FILE ST002748_AN004458_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Seconds #END