#METABOLOMICS WORKBENCH Bkaipparettu_20250102_112031 DATATRACK_ID:5502 STUDY_ID:ST003651 ANALYSIS_ID:AN005997 PROJECT_ID:PR002261 VERSION 1 CREATED_ON January 6, 2025, 10:14 pm #PROJECT PR:PROJECT_TITLE Biguanides antithetically regulate tumor properties by the dose-dependent PR:PROJECT_TITLE mitochondrial reprogramming-driven c-Src pathway PR:PROJECT_SUMMARY The biguanide metformin attenuates mitochondrial oxidation and has been proposed PR:PROJECT_SUMMARY as an anti-cancer therapy. However, recent clinical studies suggested increased PR:PROJECT_SUMMARY proliferation and fatty acid beta-oxidation (FAO) in a subgroup of breast cancer PR:PROJECT_SUMMARY (BC) patients after metformin therapy. Considering that FAO can activate Src PR:PROJECT_SUMMARY kinase in aggressive triple-negative BC (TNBC), we hypothesized that a low-dose PR:PROJECT_SUMMARY biguanides-driven AMPK-ACC-FAO signaling may activate the Src pathway in TNBC. PR:PROJECT_SUMMARY The low bioavailability of metformin in TNBC xenografts mimicked metformin's in PR:PROJECT_SUMMARY vitro low-dose effect. Pharmacological or genetic inhibition of FAO PR:PROJECT_SUMMARY significantly enhanced the anti-tumor properties of biguanides. Lower doses of PR:PROJECT_SUMMARY biguanides induced and higher doses suppressed Src signaling. Dasatinib and PR:PROJECT_SUMMARY metformin synergistically inhibited TNBC patient-derived xenograft growth but PR:PROJECT_SUMMARY not in high-fat diet-fed mice. This combination also suppressed TNBC metastatic PR:PROJECT_SUMMARY progression. A combination of biguanides with Src inhibitors provides synergy to PR:PROJECT_SUMMARY target metastatic TNBC suffering with limited treatment options. PR:INSTITUTE Baylor College of Medicine PR:LAST_NAME Kaipparettu PR:FIRST_NAME Benny PR:ADDRESS One Baylor Plaza, Houston, Texas, 77030, USA PR:EMAIL kaippare@bcm.edu PR:PHONE 7137985469 #STUDY ST:STUDY_TITLE Biguanides antithetically regulate tumor properties by the dose-dependent ST:STUDY_TITLE mitochondrial reprogramming-driven c-Src pathway ST:STUDY_SUMMARY The biguanide metformin attenuates mitochondrial oxidation and has been proposed ST:STUDY_SUMMARY as an anti-cancer therapy. However, recent clinical studies suggested increased ST:STUDY_SUMMARY proliferation and fatty acid beta-oxidation (FAO) in a subgroup of breast cancer ST:STUDY_SUMMARY (BC) patients after metformin therapy. Considering that FAO can activate Src ST:STUDY_SUMMARY kinase in aggressive triple-negative BC (TNBC), we hypothesized that a low-dose ST:STUDY_SUMMARY biguanides-driven AMPK-ACC-FAO signaling may activate the Src pathway in TNBC. ST:STUDY_SUMMARY The low bioavailability of metformin in TNBC xenografts mimicked metformin's in ST:STUDY_SUMMARY vitro low-dose effect. Pharmacological or genetic inhibition of FAO ST:STUDY_SUMMARY significantly enhanced the anti-tumor properties of biguanides. Lower doses of ST:STUDY_SUMMARY biguanides induced and higher doses suppressed Src signaling. Dasatinib and ST:STUDY_SUMMARY metformin synergistically inhibited TNBC patient-derived xenograft growth but ST:STUDY_SUMMARY not in high-fat diet-fed mice. This combination also suppressed TNBC metastatic ST:STUDY_SUMMARY progression. A combination of biguanides with Src inhibitors provides synergy to ST:STUDY_SUMMARY target metastatic TNBC suffering with limited treatment options. ST:INSTITUTE Baylor College of Medicine ST:LAST_NAME Kaipparettu ST:FIRST_NAME Benny ST:ADDRESS One Baylor Plaza, Houston, Texas, 77030, USA ST:EMAIL kaippare@bcm.edu ST:PHONE 7137985469 #SUBJECT SU:SUBJECT_TYPE Cultured cells 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 - 7-30-2021 Benny Cell lines A1 Treatment:Control | Sample source:SUM159 cell line RAW_FILE_NAME(Raw file name)=7-30-2021 Benny Cell Lines A1.d SUBJECT_SAMPLE_FACTORS - 7-30-2021 Benny Cell lines A2 Treatment:Control | Sample source:SUM159 cell line RAW_FILE_NAME(Raw file name)=7-30-2021 Benny Cell Lines A2.d SUBJECT_SAMPLE_FACTORS - 7-30-2021 Benny Cell lines A3 Treatment:Control | Sample source:SUM159 cell line RAW_FILE_NAME(Raw file name)=7-30-2021 Benny Cell Lines A3.d SUBJECT_SAMPLE_FACTORS - 7-30-2021 Benny Cell lines A4 Treatment:Control | Sample source:SUM159 cell line RAW_FILE_NAME(Raw file name)=7-30-2021 Benny Cell Lines A4.d SUBJECT_SAMPLE_FACTORS - 7-30-2021 Benny Cell lines B1 Treatment:Met 0.01 mM | Sample source:SUM159 cell line RAW_FILE_NAME(Raw file name)=7-30-2021 Benny Cell Lines B1.d SUBJECT_SAMPLE_FACTORS - 7-30-2021 Benny Cell lines B2 Treatment:Met 0.01 mM | Sample source:SUM159 cell line RAW_FILE_NAME(Raw file name)=7-30-2021 Benny Cell Lines B2.d SUBJECT_SAMPLE_FACTORS - 7-30-2021 Benny Cell lines B3 Treatment:Met 0.01 mM | Sample source:SUM159 cell line RAW_FILE_NAME(Raw file name)=7-30-2021 Benny Cell Lines B3.d SUBJECT_SAMPLE_FACTORS - 7-30-2021 Benny Cell lines B4 Treatment:Met 0.01 mM | Sample source:SUM159 cell line RAW_FILE_NAME(Raw file name)=7-30-2021 Benny Cell Lines B4.d SUBJECT_SAMPLE_FACTORS - 7-30-2021 Benny Cell lines C1 Treatment:Met 0.05 mM | Sample source:SUM159 cell line RAW_FILE_NAME(Raw file name)=7-30-2021 Benny Cell Lines C1.d SUBJECT_SAMPLE_FACTORS - 7-30-2021 Benny Cell lines C2 Treatment:Met 0.05 mM | Sample source:SUM159 cell line RAW_FILE_NAME(Raw file name)=7-30-2021 Benny Cell Lines C2.d SUBJECT_SAMPLE_FACTORS - 7-30-2021 Benny Cell lines C3 Treatment:Met 0.05 mM | Sample source:SUM159 cell line RAW_FILE_NAME(Raw file name)=7-30-2021 Benny Cell Lines C3.d SUBJECT_SAMPLE_FACTORS - 7-30-2021 Benny Cell lines C4 Treatment:Met 0.05 mM | Sample source:SUM159 cell line RAW_FILE_NAME(Raw file name)=7-30-2021 Benny Cell Lines C4.d SUBJECT_SAMPLE_FACTORS - 7-30-2021 Benny Cell lines D1 Treatment:Met 5 mM | Sample source:SUM159 cell line RAW_FILE_NAME(Raw file name)=7-30-2021 Benny Cell Lines D1.d SUBJECT_SAMPLE_FACTORS - 7-30-2021 Benny Cell lines D2 Treatment:Met 5 mM | Sample source:SUM159 cell line RAW_FILE_NAME(Raw file name)=7-30-2021 Benny Cell Lines D2.d SUBJECT_SAMPLE_FACTORS - 7-30-2021 Benny Cell lines D3 Treatment:Met 5 mM | Sample source:SUM159 cell line RAW_FILE_NAME(Raw file name)=7-30-2021 Benny Cell Lines D3.d SUBJECT_SAMPLE_FACTORS - 7-30-2021 Benny Cell lines D4 Treatment:Met 5 mM | Sample source:SUM159 cell line RAW_FILE_NAME(Raw file name)=7-30-2021 Benny Cell Lines D4.d #COLLECTION CO:COLLECTION_SUMMARY SUM-159 is a breast cancer cell line CO:COLLECTION_SUMMARY (https://www.cellosaurus.org/pawefish/BreastCellLineDescriptions/SUM-159.html). CO:COLLECTION_SUMMARY The Metformin treated SUM-159 cells were washed with PBS followed by pellet CO:COLLECTION_SUMMARY collection using centrifugation. The pellet was submitted to metabolomics core. CO:COLLECTION_SUMMARY (3*10^6 cells per pellet). CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY In the treatment groups (Metformin 0.01 mM, Metformin 0.05 mM, and Metformin 5 TR:TREATMENT_SUMMARY mM), Metformin was administered with a concentration of 0.01, 0.05 and 5 mM TR:TREATMENT_SUMMARY respectively for 24 hours. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The cells were freeze thawed at 3 times in liquid nitrogen. The extraction step SP:SAMPLEPREP_SUMMARY starts with addition of 750 μL ice-cold methanol:water (4:1) containing 20 μL SP:SAMPLEPREP_SUMMARY spiked internal standards (ISTD). After homogenization, ice-cold chloroform and SP:SAMPLEPREP_SUMMARY water were added in a 3:1 ratio for a final proportion of 4:3:2 SP:SAMPLEPREP_SUMMARY methanol:chloroform:water. The organic and aqueous layers were collected, dried, SP:SAMPLEPREP_SUMMARY and resuspended in methanol:water (1:1). The extract was deproteinized using a SP:SAMPLEPREP_SUMMARY 3-kDa molecular filter and the filtrate was dried under vacuum. The dried SP:SAMPLEPREP_SUMMARY extracts were resuspended in 100 μL of injection solvent composed of 1:1 SP:SAMPLEPREP_SUMMARY methanol:water and subjected to LC-MS. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The extracted amino acids and carnitines were separated through the ZORBAX CH:CHROMATOGRAPHY_SUMMARY Eclipse XDB C-18 HPLC column via positive ionization mode in MS. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity CH:COLUMN_NAME Agilent ZORBAX Eclipse XDB-C18 (50 x 4.6mm,1.8um) CH:SOLVENT_A 100% Water; 0.1% formic acid CH:SOLVENT_B 100% Acetonitrile; 0.1% formic acid CH:FLOW_GRADIENT 0-5 min 30% B, 5-15 min 70% B, 15-20 min 90% B, and 20- 20.10 min 30% B, CH:FLOW_GRADIENT followed by re-equilibration till the end of the gradient 25 min to the initial CH:FLOW_GRADIENT starting condition of 30% B. CH:FLOW_RATE 0.2 mL/min CH:COLUMN_TEMPERATURE 37 CH:INJECTION_TEMPERATURE 37 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6495 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Mass spectrometry data were acquired via multiple reaction monitoring (MRM) MS:MS_COMMENTS using a 6495 Triple Quadrupole mass spectrometry coupled to an HPLC system MS:MS_COMMENTS (Agilent Technologies, Santa Clara, CA) Agilent Mass Hunter Software. The peak MS:MS_COMMENTS integration and data analysis were performed using Agilent Mass Hunter MS:MS_COMMENTS Quantitative Analysis software. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak Area MS_METABOLITE_DATA_START Samples 7-30-2021 Benny Cell lines A1 7-30-2021 Benny Cell lines A2 7-30-2021 Benny Cell lines A3 7-30-2021 Benny Cell lines A4 7-30-2021 Benny Cell lines B1 7-30-2021 Benny Cell lines B2 7-30-2021 Benny Cell lines B3 7-30-2021 Benny Cell lines B4 7-30-2021 Benny Cell lines C1 7-30-2021 Benny Cell lines C2 7-30-2021 Benny Cell lines C3 7-30-2021 Benny Cell lines C4 7-30-2021 Benny Cell lines D1 7-30-2021 Benny Cell lines D2 7-30-2021 Benny Cell lines D3 7-30-2021 Benny Cell lines D4 Factors Treatment:Control | Sample source:SUM159 cell line Treatment:Control | Sample source:SUM159 cell line Treatment:Control | Sample source:SUM159 cell line Treatment:Control | Sample source:SUM159 cell line Treatment:Met 0.01 mM | Sample source:SUM159 cell line Treatment:Met 0.01 mM | Sample source:SUM159 cell line Treatment:Met 0.01 mM | Sample source:SUM159 cell line Treatment:Met 0.01 mM | Sample source:SUM159 cell line Treatment:Met 0.05 mM | Sample source:SUM159 cell line Treatment:Met 0.05 mM | Sample source:SUM159 cell line Treatment:Met 0.05 mM | Sample source:SUM159 cell line Treatment:Met 0.05 mM | Sample source:SUM159 cell line Treatment:Met 5 mM | Sample source:SUM159 cell line Treatment:Met 5 mM | Sample source:SUM159 cell line Treatment:Met 5 mM | Sample source:SUM159 cell line Treatment:Met 5 mM | Sample source:SUM159 cell line L-Tryptophan (ISTD) 6104158 4958735 4546356 5515840 5381765 5649255 5122592 5467960 5928682 5268568 5099651 5468930 5059699 6157147 5572547 5494978 Deoxy carnitine 52889 50795 54426 55730 49318 57689 51410 61702 90410 96562 92528 110064 39292 39395 42456 39439 Carnitine 117183 122799 121764 120510 123321 114874 132667 133460 134440 136117 142170 176682 58907 51723 55148 51496 Acetyl Carnitine 8700003 9086827 8452604 9234448 8593944 8980911 9254466 9590174 10491059 10170758 10547154 11749133 4319796 4050824 3906573 4288565 Propeonyl Carnitine 5796067 5480931 4451094 5606198 4838752 5070518 4404175 5233438 4859154 4861092 4532430 4337147 1288926 1538217 1497939 1438081 2-Methyl butyryl carnitine/Isovalery carnitine 2409081 2217618 1555453 2389300 2019969 2133199 1610059 2066630 2312683 2320571 2073818 2141235 849976 1212921 1067536 1027950 Glutaryl carnitine 60616 62882 54932 64913 61615 63613 70431 66195 66082 68650 59482 58967 112082 142304 151548 119607 Butryl Carnitine 25368078 25566396 18206993 27776582 24204245 28464995 19675347 26674751 28319544 26804793 27902955 26121059 11844066 15330242 15341937 14287482 Isobutyryl Carnitine 21609459 20299846 14675155 22064309 19712004 21134813 15540708 20825848 21840566 20966114 20827616 20130654 8887006 11297813 11190803 10522576 Malonylcarnitine 13144 14837 15935 16070 14680 17336 15956 12601 18766 17815 15801 14442 15751 18132 24593 20689 Hexanoyl carnitine 1459046 1624260 1286680 1605959 1496431 1655517 1409700 1674571 1842516 1851531 1896845 1593250 1125394 1271979 1255106 1029316 MethylGlutaryl carnitine 12681 13663 12045 15768 11383 14284 11392 12072 15054 14758 15721 11519 15758 14371 15667 14167 Octonyl Carnitine 716532 836109 706203 898507 676012 702978 641480 662195 691323 811554 762497 677855 1064485 987844 1074847 909691 Decanoyl carnitine 272770 317573 263620 320694 293395 274701 266592 263189 314375 388882 359840 309690 214124 199263 241037 173202 Lauroyl carnitine 476350 496841 427794 479196 429603 454065 418206 366679 517522 559087 522382 469799 299317 288192 319809 270119 Myristoyl carnitine 1369646 1768463 1696957 1721499 1521319 1681391 1812879 1390448 1980980 2118274 1913490 1790679 1645463 1524024 1574520 1732955 Palmitoylcarnitine 306356 348892 425044 395587 340985 473063 528198 325398 519969 521814 491821 480042 445718 352171 440524 604912 Stearoyl carnitine 16493 14777 14567 18843 18180 20113 19063 20835 30098 21970 34758 29193 19442 19064 18548 18181 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name L-Tryptophan (ISTD) Deoxy carnitine Carnitine Acetyl Carnitine Propeonyl Carnitine 2-Methyl butyryl carnitine/Isovalery carnitine Glutaryl carnitine Butryl Carnitine Isobutyryl Carnitine Malonylcarnitine Hexanoyl carnitine MethylGlutaryl carnitine Octonyl Carnitine Decanoyl carnitine Lauroyl carnitine Myristoyl carnitine Palmitoylcarnitine Stearoyl carnitine METABOLITES_END #END