#METABOLOMICS WORKBENCH zhaoyuan0315_20241104_222044 DATATRACK_ID:5345 STUDY_ID:ST003569 ANALYSIS_ID:AN005863 PROJECT_ID:PR002202 VERSION 1 CREATED_ON November 12, 2024, 9:41 am #PROJECT PR:PROJECT_TITLE High mobility group A1 (HMGA1) promotes the tumorigenesis of colorectal cancer PR:PROJECT_TITLE by increasing lipid synthesis PR:PROJECT_TYPE LC-MS PR:PROJECT_SUMMARY Metabolic reprogramming is a hallmark of cancer, enabling tumor cells to meet PR:PROJECT_SUMMARY the high energy and biosynthetic demands required for their proliferation. High PR:PROJECT_SUMMARY mobility group A1 (HMGA1) is a structural transcription factor and frequently PR:PROJECT_SUMMARY overexpressed in human colorectal cancer (CRC). Here, we show that HMGA1 PR:PROJECT_SUMMARY promotes CRC progression by driving lipid synthesis in a AOM/DSS-induced CRC PR:PROJECT_SUMMARY mouse model. Using conditional knockout (Hmga1△IEC) and knock-in PR:PROJECT_SUMMARY (Hmga1IEC-OE/+) mouse models, we demonstrate that HMGA1 enhances CRC cell PR:PROJECT_SUMMARY proliferation and accelerates tumor development by upregulating fatty acid PR:PROJECT_SUMMARY synthase (FASN). Mechanistically, HMGA1 increases the transcriptional activity PR:PROJECT_SUMMARY of sterol regulatory element-binding protein 1 (SREBP1) on the FASN promoter, PR:PROJECT_SUMMARY leading to increased lipid accumulation in intestinal epithelial cells. PR:PROJECT_SUMMARY Moreover, a high-fat diet exacerbates CRC progression in Hmga1△IEC mice, while PR:PROJECT_SUMMARY pharmacological inhibition of FASN by orlistat reduces tumor growth in PR:PROJECT_SUMMARY Hmga1IEC-OE/+ mice. Our findings suggest that targeting lipid metabolism could PR:PROJECT_SUMMARY offer a promising therapeutic strategy for CRC. PR:INSTITUTE Henan University PR:LAST_NAME Xu PR:FIRST_NAME Zhi-Xiang PR:ADDRESS Jinming Road, Kaifeng, Henan province, 475000, China PR:EMAIL zhixiangxu08@gmail.com PR:PHONE 86-13270538760 #STUDY ST:STUDY_TITLE Differentially regulated metabolites in colorectal cancer from Rosa26Hmga1/+ and ST:STUDY_TITLE Hmga1IEC-OE/+mice ST:STUDY_SUMMARY Dysregulations of cell metabolism, such as elevated aerobic glycolysis and ST:STUDY_SUMMARY increased fatty acid metabolism, play a critical role in the tumorigenesis of ST:STUDY_SUMMARY colorectal cancer (CRC). To determine whether HMGA1 promotes CRC through ST:STUDY_SUMMARY regulating cell metabolism, we performed an untargeted metabolomics using ST:STUDY_SUMMARY intestinal epithelial cells (IECs) from Hmga1flox/flox and Hmga1△IEC mice ST:STUDY_SUMMARY firstly. The results showed that fatty acyl metabolites were mainly ST:STUDY_SUMMARY down-regulated in HMGA1-deficient IECs. Further, untargeted metabolomics ST:STUDY_SUMMARY analysis on tumors derived from the Hmga1IEC-OE/+ mice also showed that fatty ST:STUDY_SUMMARY acyl metabolites were mainly up-regulated by HMGA1 overexpression. FA is a major ST:STUDY_SUMMARY component of structurally complex lipids and is one of the most basic categories ST:STUDY_SUMMARY of bio-lipids. We analyzed all lipids-related metabolites and found the free ST:STUDY_SUMMARY fatty acids were significantly up-regulated in tumors derived from the ST:STUDY_SUMMARY Hmga1IEC-OE/+ mice. Thus, untargeted metabolomics measurement of IECs (CRCs) ST:STUDY_SUMMARY from both Hmga1 conditional knockout (Hmga1△IEC) and knock-in (Hmga1IEC-OE/+) ST:STUDY_SUMMARY mice demonstrates a positive correlation between expression of HMGA1 and ST:STUDY_SUMMARY activation of fatty acid synthesis. ST:INSTITUTE Henan University ST:LAST_NAME Xu ST:FIRST_NAME Zhi-Xiang ST:ADDRESS Jinming Road, Kaifeng, Henan province, 475000, China ST:EMAIL zhixiangxu08@gmail.com ST:PHONE 86-13270538760 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #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 Rosa26Hmga1/+-1 T23320676a Sample source:Colorectum tissue | Genotype:Wild-type RAW_FILE_NAME=T23320676a SUBJECT_SAMPLE_FACTORS Rosa26Hmga1/+-2 T23320679a Sample source:Colorectum tissue | Genotype:Wild-type RAW_FILE_NAME=T23320679a SUBJECT_SAMPLE_FACTORS Rosa26Hmga1/+-3 T23320682a Sample source:Colorectum tissue | Genotype:Wild-type RAW_FILE_NAME=T23320682a SUBJECT_SAMPLE_FACTORS Hmga1IEC-OE/+-1 T23320685a Sample source:Colorectum tissue | Genotype:HMGA1-knockin RAW_FILE_NAME=T23320685a SUBJECT_SAMPLE_FACTORS Hmga1IEC-OE/+-2 T23320688a Sample source:Colorectum tissue | Genotype:HMGA1-knockin RAW_FILE_NAME=T23320688a SUBJECT_SAMPLE_FACTORS Hmga1IEC-OE/+-3 T23320691a Sample source:Colorectum tissue | Genotype:HMGA1-knockin RAW_FILE_NAME=T23320691a #COLLECTION CO:COLLECTION_SUMMARY The C57BL/6J mice were selected for the induction of CRC using CO:COLLECTION_SUMMARY azoxymethane/dextran sulfate sodium (AOM/DSS). Mice received an intraperitoneal CO:COLLECTION_SUMMARY injection of AOM (Sigma-Aldrich, St. Louis, MO, USA) at 10 mg/kg body weight. CO:COLLECTION_SUMMARY One week later, they were given 2% (w/v) DSS (Meilunbio, Dalian, China) in CO:COLLECTION_SUMMARY drinking water for 5 days, followed by regular water for 14 days. This sequence, CO:COLLECTION_SUMMARY termed a DSS cycle, was repeated three times. Body weight and stool were CO:COLLECTION_SUMMARY continuously monitored until euthanasia at week 12. Since orthotopic colorectal CO:COLLECTION_SUMMARY tumors induced by AOM/DSS were not directly visible during the mice's lifetime, CO:COLLECTION_SUMMARY we determined the timing of sacrifice based on weight loss and behavioral CO:COLLECTION_SUMMARY observations. Mice were euthanized when they lost 20% of their pre-experiment CO:COLLECTION_SUMMARY body weight, showed signs of severe debilitation, or were on the verge of death, CO:COLLECTION_SUMMARY such as being unable to move, and/or body condition scoring (BCS) reached 2.0. CO:COLLECTION_SUMMARY Distal colon tissues were collected, and visible tumors on the colorectal mucosa CO:COLLECTION_SUMMARY were counted. The samples was saved at -80℃ condition. CO:SAMPLE_TYPE Colorectum #TREATMENT TR:TREATMENT_SUMMARY The samples were not subjected to any further treatment. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The sample was thawed on ice and homogenized by a grinder (30 HZ) for 20 s. A SP:SAMPLEPREP_SUMMARY 400 μL solution (Methanol: Water = 7:3, V/V) containing internal standard was SP:SAMPLEPREP_SUMMARY added in to 20 mg ground sample, and shaken at 1500 rpm for 5 min. After placing SP:SAMPLEPREP_SUMMARY on ice for 15 min, the sample was centrifuged at 12000 rpm for 10 min (4 °C). A SP:SAMPLEPREP_SUMMARY 300 μL of supernatant was collected and placed in -20 °C for 30 min. The SP:SAMPLEPREP_SUMMARY sample was then centrifuged at 12000 rpm for 3 min (4 °C). A 200 μL aliquots SP:SAMPLEPREP_SUMMARY of supernatant were transferred for LC-MS analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Shimadzu Nexera LC-30A CH:COLUMN_NAME Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile; 0.1% formic acid CH:FLOW_GRADIENT A/B (95:5, V/V) at 0 min, 80:20 V/V at 2.0 min, 40:60 V/V at 5 min, 1:99 V/V at CH:FLOW_GRADIENT 6 min, 1:99 V/V at 7.5 min, 95: 5 V/V at 7.6 min, 95: 5 V/V at 10 min CH:FLOW_RATE 0.4 mL/min CH:COLUMN_TEMPERATURE 40 °C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME ABI Sciex 6600 TripleTOF MS:INSTRUMENT_TYPE Triple TOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The data acquisition was operated using the information-dependent acquisition MS:MS_COMMENTS (IDA) mode using Analyst TF 1.7.1 Software (Sciex, Concord, ON, Canada). The MS:MS_COMMENTS source parameters were set as follows: ion source gas 1 (GAS1),50 psi; ion MS:MS_COMMENTS source gas 2 (GAS2), 50 psi; curtain gas (CUR), 25 psi; temperature(TEM), 550 MS:MS_COMMENTS °C; declustering potential (DP), 60 V, or−60 V in positive or negative modes, MS:MS_COMMENTS respectively; and ion spray voltagefloating (ISVF), 5000 V or−4000 V in MS:MS_COMMENTS positive or negative modes, respectively. The TOF MS scan parameters were set as MS:MS_COMMENTS follows: mass range, 50–1000 Da; accumulation time, 200 ms; and dynamic MS:MS_COMMENTS background subtract, on. The product ion scan parameters were set as follows: MS:MS_COMMENTS mass range, 25–1000 Da; accumulation time, 40 ms; collision energy, 30 or−30 MS:MS_COMMENTS V in positive or negative modes, respectively; collision energy spread, 15; MS:MS_COMMENTS resolution, UNIT; charge state, 1 to 1; intensity, 100 cps; exclude isotopes MS:MS_COMMENTS within 4 Da; mass tolerance, 50 ppm; maximum number of candidate ions to monitor MS:MS_COMMENTS per cycle, 18. MS:MS_RESULTS_FILE ST003569_AN005863_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END