{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002274","ANALYSIS_ID":"AN003716","VERSION":"1","CREATED_ON":"September 1, 2022, 10:56 am"},

"PROJECT":{"PROJECT_TITLE":"1-deoxysphingolipid synthesis compromises anchorage-independent growth and plasma membrane endocytosis in cancer cells","PROJECT_TYPE":"Manuscript","PROJECT_SUMMARY":"Serine palmitoyltransferase (SPT) predominantly incorporates serine and fatty acyl-CoAs into diverse sphingolipids that serve as structural components of membranes and signaling molecules within or amongst cells. However, SPT also uses alanine as a substrate in the contexts of low serine availability, alanine accumulation, or disease-causing mutations in hereditary sensory neuropathy type I (HSAN1), resulting in the synthesis and accumulation of 1-deoxysphingolipids. These species promote cytotoxicity in neurons and impact diverse cellular phenotypes, including suppression of anchorage-independent cancer cell growth. While altered serine and alanine can promote 1-deoxysphingolipid synthesis, they impact numerous other metabolic pathways important for cancer cells. Here we combined isotope tracing, quantitative metabolomics, and functional studies to better understand the mechanistic drivers of 1-deoxysphingolipid toxicity in cancer cells. Both alanine treatment and SPTLC1C133W expression induce 1-deoxy(dihydro)ceramide synthesis and accumulation but fail to broadly impact intermediary metabolism, abundances of other lipids, or growth of adherent cells. However, spheroid culture and soft agar colony formation were compromised when endogenous 1-deoxysphingolipid synthesis was induced via SPTLC1C133W expression. Consistent with these impacts on anchorage-independent cell growth, we observed that 1-deoxysphingolipid synthesis reduced plasma membrane endocytosis. These results highlight a potential role for SPT promiscuity in linking altered amino acid metabolism to plasma membrane endocytosis.","INSTITUTE":"TU Braunschweig","LAST_NAME":"Cordes","FIRST_NAME":"Thekla","ADDRESS":"Rebenring 56, Braunschweig, Lower Saxony, 38106, Germany","EMAIL":"thekla.cordes@tu-bs.de","PHONE":"04953139155202","FUNDING_SOURCE":"NIH NCI R01CA234245 and R50CA252146","PUBLICATIONS":"JLR accepted","CONTRIBUTORS":"Thekla Cordes (University of California San Diego; Salk Institute for Biological Studies; TU Braunschweig), Christian Metallo (University of California, San Diego; Salk Institute for Biological Studies)"},

"STUDY":{"STUDY_TITLE":"1-deoxysphingolipid synthesis compromises anchorage-independent growth and plasma membrane endocytosis in cancer cells","STUDY_SUMMARY":"Serine palmitoyltransferase (SPT) predominantly incorporates serine and fatty acyl-CoAs into diverse sphingolipids that serve as structural components of membranes and signaling molecules within or amongst cells. However, SPT also uses alanine as a substrate in the contexts of low serine availability, alanine accumulation, or diseasecausing mutations in hereditary sensory neuropathy type I (HSAN1), resulting in the synthesis and accumulation of 1-deoxysphingolipids. These species promote cytotoxicity in neurons and impact diverse cellular phenotypes, including suppression of anchorage-independent cancer cell growth. While altered serine and alanine can promote 1-deoxysphingolipid synthesis, they impact numerous other metabolic pathways important for cancer cells. Here we combined isotope tracing, quantitative metabolomics, and functional studies to better understand the mechanistic drivers of 1- deoxysphingolipid toxicity in cancer cells. Both alanine treatment and SPTLC1 C133W expression induce 1-deoxy(dihydro)ceramide synthesis and accumulation but fail to broadly impact intermediary metabolism, abundances of other lipids, or growth of adherent cells. However, spheroid culture and soft agar colony formation were compromised when endogenous 1-deoxysphingolipid synthesis was induced via SPTLC1 C133W expression. Consistent with these impacts on anchorageindependent cell growth, we observed that 1-deoxysphingolipid synthesis reduced plasma membrane endocytosis. These results highlight a potential role for SPT promiscuity in linking altered amino acid metabolism to plasma membrane endocytosis.","INSTITUTE":"Salk Institute for Biological Studies","LABORATORY":"Molecular and Cell Biology Laboratory (Christian Metallo)","LAST_NAME":"Cordes","FIRST_NAME":"Thekla","ADDRESS":"10010 N Torrey Pines Rd, La Jolla, CA 92037, United States","EMAIL":"thekla.cordes@tu-bs.de","PHONE":"004953139155202"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606","CELL_BIOSOURCE_OR_SUPPLIER":"ATCC, CCL-247","CELL_STRAIN_DETAILS":"HCT116"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"SPT_WT_15min_0131111",
"Factors":{"Genotype":"Wild-type","Treatment":"15min"},
"Additional sample data":{"RAW_FILE_NAME":"SPT_WT_15min_0131111.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"SPT_WT_15min_0131112",
"Factors":{"Genotype":"Wild-type","Treatment":"15min"},
"Additional sample data":{"RAW_FILE_NAME":"SPT_WT_15min_0131112.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"SPT_WT_15min_0131113",
"Factors":{"Genotype":"Wild-type","Treatment":"15min"},
"Additional sample data":{"RAW_FILE_NAME":"SPT_WT_15min_0131113.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"SPT_WT_1h_0131114",
"Factors":{"Genotype":"Wild-type","Treatment":"1h"},
"Additional sample data":{"RAW_FILE_NAME":"SPT_WT_1h_0131114.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"SPT_WT_1h_0131115",
"Factors":{"Genotype":"Wild-type","Treatment":"1h"},
"Additional sample data":{"RAW_FILE_NAME":"SPT_WT_1h_0131115.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"SPT_WT_1h_0131116",
"Factors":{"Genotype":"Wild-type","Treatment":"1h"},
"Additional sample data":{"RAW_FILE_NAME":"SPT_WT_1h_0131116.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"SPT_WT_4h_0131117",
"Factors":{"Genotype":"Wild-type","Treatment":"4h"},
"Additional sample data":{"RAW_FILE_NAME":"SPT_WT_4h_0131117.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"SPT_WT_4h_0131118",
"Factors":{"Genotype":"Wild-type","Treatment":"4h"},
"Additional sample data":{"RAW_FILE_NAME":"SPT_WT_4h_0131118.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"SPT_WT_4h_0131119",
"Factors":{"Genotype":"Wild-type","Treatment":"4h"},
"Additional sample data":{"RAW_FILE_NAME":"SPT_WT_4h_0131119.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"SPT_C133W_15min_01311110",
"Factors":{"Genotype":"C133W","Treatment":"15min"},
"Additional sample data":{"RAW_FILE_NAME":"SPT_C133W_15min_01311110.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"SPT_C133W_15min_01311111",
"Factors":{"Genotype":"C133W","Treatment":"15min"},
"Additional sample data":{"RAW_FILE_NAME":"SPT_C133W_15min_01311111.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"SPT_C133W_15min_01311112",
"Factors":{"Genotype":"C133W","Treatment":"15min"},
"Additional sample data":{"RAW_FILE_NAME":"SPT_C133W_15min_01311112.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"SPT_C133W_1h_01311113",
"Factors":{"Genotype":"C133W","Treatment":"1h"},
"Additional sample data":{"RAW_FILE_NAME":"SPT_C133W_1h_01311113.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"SPT_C133W_1h_01311114",
"Factors":{"Genotype":"C133W","Treatment":"1h"},
"Additional sample data":{"RAW_FILE_NAME":"SPT_C133W_1h_01311114.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"SPT_C133W_1h_01311115",
"Factors":{"Genotype":"C133W","Treatment":"1h"},
"Additional sample data":{"RAW_FILE_NAME":"SPT_C133W_1h_01311115.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"SPT_C133W_4h_01311116",
"Factors":{"Genotype":"C133W","Treatment":"4h"},
"Additional sample data":{"RAW_FILE_NAME":"SPT_C133W_4h_01311116.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"SPT_C133W_4h_01311117",
"Factors":{"Genotype":"C133W","Treatment":"4h"},
"Additional sample data":{"RAW_FILE_NAME":"SPT_C133W_4h_01311117.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"SPT_C133W_4h_01311118",
"Factors":{"Genotype":"C133W","Treatment":"4h"},
"Additional sample data":{"RAW_FILE_NAME":"SPT_C133W_4h_01311118.mzXML"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"For tracing studies with [U-13C16]palmitate, HCT116 cells were cultured in growth medium in the presence of 0.1 μg/ml doxycycline for 7 days before tracer start. Growth medium was replaced to DMEM medium containing 1% (v/v) delipidated FBS 24h prior tracer start and medium exchange again 1h prior tracer trace. [U-13C16]palmitate was noncovalently bound to fatty acid-free BSA and added to culture medium at 5% of the final volume (50 μM final concentration). Media was prewarmed to 37oC in a cell incubator with 5% CO2 and cells were traced for 15 min, 1 h, and 4 h. For targeted sphingolipid analysis, cells were washed with 0.9% (w/v) NaCl and extracted with 0.25 mL of −20°C methanol, 0.25 mL -20°C chloroform, and 0.1 mL of water. The tubes were vortexed for 5 min, centrifuged at 20,000 x g at 4°C for 5 min, and the lower organic phase was collected. The remaining polar phase was extracted with 2 μL formic acid and 0.25 mL of -20 °C chloroform. The organic phases were combined, dried under air, resuspended in 80 μL Buffer B (0.2% formic acid and 1 mM ammonium formate in methanol), sonicated for 10 min, and centrifuged for 10 min at 20,000 x g at 4 °C. The supernatant was transferred to LC vials containing glass inserts for analysis on a Q-Exactive LC-MS system.","SAMPLE_TYPE":"Cultured cells"},

"TREATMENT":{"TREATMENT_SUMMARY":"HCT116 expressing SPTLC1WT or SPTLC1C133W were cultured in media containing [U-13C16]palmitate for 15min, 1h, and 4h."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"The samples were reconstituted in 80 μL Buffer B (0.2% formic acid and 1 mM ammonium formate in methanol), sonicated for 10 min, and centrifuged for 10 min at 20,000 x g at 4 °C. The supernatant was transferred to LC vials containing glass inserts for analysis on a Q-Exactive LC-MS system."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Thermo Vanquish","COLUMN_NAME":"Kinetex 2.6 μM C8 100 Å 150 x 3 mm LC column (Phenomenex)"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS","LABORATORY_NAME":"Christian Metallo"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"To quantify labeling on SL and deoxySL species from [U-13C16]palmitate a Q Exactive orbitrap mass spectrometer with a Vanquish Flex Binary UHPLC system (Thermo Scientific) was used with a Kinetex 2.6 μM C8 100 Å 150 x 3 mm LC column (Phenomenex) at 40°C. 5 μL of sample was injected. Chromatography was performed using a gradient of 2 mM ammonium formate and 0.2 % formic acid (mobile phase A) and 1 mM ammonium formate and 0.2 % formic acid in methanol (mobile phase B), at a flow rate of 0.5 mL/min. The LC gradient held at 82% B for 0-3 min, then ran from 82%-90% B in 3-4 min, then 90-99% in 4-18 min, held at 99% B for 7 min, then reduced from 99%-82% from 25-27 min, then held at 82% for a further 13 mins. Lipids were analyzed in positive mode using spray voltage 3 kV. Sweep gas flow was 5 arbitrary units, auxiliary gas flow 7 arbitrary units and sheath gas flow 50 arbitrary units, with a capillary temperature of 300°C. Full MS (scan range 150-2000 m/z) was used at 70 000 resolution with 1e6 automatic gain control and a maximum injection time of 200 ms. Data dependent MS2 (Top 6) mode at 17 500 resolution with automatic gain control set at 1e5 with a maximum injection time of 50 ms was used for peak identification, combined with known standards where possible."},

"MS_METABOLITE_DATA":{
"Units":"ion counts",

"Data":[{"Metabolite":"Cer d18:1/24:1","SPT_C133W_15min_01311110":"12151155.54","SPT_C133W_15min_01311111":"10205801.85","SPT_C133W_15min_01311112":"8119015.47","SPT_C133W_1h_01311113":"8740968.8","SPT_C133W_1h_01311114":"10892397.36","SPT_C133W_1h_01311115":"15546699.37","SPT_C133W_4h_01311116":"12183738.41","SPT_C133W_4h_01311117":"12657917.77","SPT_C133W_4h_01311118":"18556726.2","SPT_WT_15min_0131111":"6894899.97","SPT_WT_15min_0131112":"9097543.84","SPT_WT_15min_0131113":"9707655.69","SPT_WT_1h_0131114":"17412002.16","SPT_WT_1h_0131115":"12209404.72","SPT_WT_1h_0131116":"13858027.47","SPT_WT_4h_0131117":"12063002.8","SPT_WT_4h_0131118":"11806994.75","SPT_WT_4h_0131119":"16937500.01"},{"Metabolite":"Cer d18:1/24:0","SPT_C133W_15min_01311110":"7101466.47","SPT_C133W_15min_01311111":"5577524.07","SPT_C133W_15min_01311112":"4432532.4","SPT_C133W_1h_01311113":"4698830.23","SPT_C133W_1h_01311114":"6376552.03","SPT_C133W_1h_01311115":"14126756","SPT_C133W_4h_01311116":"6879697.68","SPT_C133W_4h_01311117":"7353515.96","SPT_C133W_4h_01311118":"10070106.88","SPT_WT_15min_0131111":"3853669.42","SPT_WT_15min_0131112":"5582265.38","SPT_WT_15min_0131113":"5752534.56","SPT_WT_1h_0131114":"10445284.89","SPT_WT_1h_0131115":"7196665.85","SPT_WT_1h_0131116":"7380665.68","SPT_WT_4h_0131117":"7220515.56","SPT_WT_4h_0131118":"6381753.28","SPT_WT_4h_0131119":"9152040.07"},{"Metabolite":"Cer d18:1/16:0","SPT_C133W_15min_01311110":"6830905.75","SPT_C133W_15min_01311111":"5798724.12","SPT_C133W_15min_01311112":"4213409.12","SPT_C133W_1h_01311113":"7502301.32","SPT_C133W_1h_01311114":"8367183.07","SPT_C133W_1h_01311115":"15262343.15","SPT_C133W_4h_01311116":"10244717.43","SPT_C133W_4h_01311117":"12104078.65","SPT_C133W_4h_01311118":"15641062.11","SPT_WT_15min_0131111":"3568996.72","SPT_WT_15min_0131112":"5195217.75","SPT_WT_15min_0131113":"6178559.37","SPT_WT_1h_0131114":"15647106.76","SPT_WT_1h_0131115":"11124028.25","SPT_WT_1h_0131116":"11924427.94","SPT_WT_4h_0131117":"11680062.15","SPT_WT_4h_0131118":"10332946.87","SPT_WT_4h_0131119":"15824413.31"},{"Metabolite":"DHCer d18:0/16:0","SPT_C133W_15min_01311110":"22143391.7","SPT_C133W_15min_01311111":"13683684.41","SPT_C133W_15min_01311112":"11902550.8","SPT_C133W_1h_01311113":"25521948.28","SPT_C133W_1h_01311114":"42804508.34","SPT_C133W_1h_01311115":"20244795.8","SPT_C133W_4h_01311116":"14332535","SPT_C133W_4h_01311117":"14668814.13","SPT_C133W_4h_01311118":"27568758.85","SPT_WT_15min_0131111":"12509405.84","SPT_WT_15min_0131112":"15779144.91","SPT_WT_15min_0131113":"19049310.39","SPT_WT_1h_0131114":"83038917.05","SPT_WT_1h_0131115":"54616598.64","SPT_WT_1h_0131116":"66879766.37","SPT_WT_4h_0131117":"8643847.96","SPT_WT_4h_0131118":"7897394.81","SPT_WT_4h_0131119":"24867557.38"},{"Metabolite":"DHCer d18:0/24:1","SPT_C133W_15min_01311110":"10448464.91","SPT_C133W_15min_01311111":"9268438.72","SPT_C133W_15min_01311112":"7853127.85","SPT_C133W_1h_01311113":"10089404.89","SPT_C133W_1h_01311114":"13007588.25","SPT_C133W_1h_01311115":"16109414.77","SPT_C133W_4h_01311116":"8624040.76","SPT_C133W_4h_01311117":"9767746.02","SPT_C133W_4h_01311118":"13622650.75","SPT_WT_15min_0131111":"5822165.67","SPT_WT_15min_0131112":"8011510.11","SPT_WT_15min_0131113":"10128684.46","SPT_WT_1h_0131114":"24503447.77","SPT_WT_1h_0131115":"15607295.47","SPT_WT_1h_0131116":"21391100.35","SPT_WT_4h_0131117":"7333889.78","SPT_WT_4h_0131118":"8990329.22","SPT_WT_4h_0131119":"11652897.29"},{"Metabolite":"DHCer d18:0/24:0","SPT_C133W_15min_01311110":"3709621.98","SPT_C133W_15min_01311111":"3395960.05","SPT_C133W_15min_01311112":"2847665.38","SPT_C133W_1h_01311113":"3299140.61","SPT_C133W_1h_01311114":"4752275.78","SPT_C133W_1h_01311115":"11464046.7","SPT_C133W_4h_01311116":"3636223.26","SPT_C133W_4h_01311117":"4243709.45","SPT_C133W_4h_01311118":"5891759.15","SPT_WT_15min_0131111":"2348186.72","SPT_WT_15min_0131112":"2633300.84","SPT_WT_15min_0131113":"3691289.04","SPT_WT_1h_0131114":"8232248.05","SPT_WT_1h_0131115":"5514211.93","SPT_WT_1h_0131116":"7122051.07","SPT_WT_4h_0131117":"3609123.2","SPT_WT_4h_0131118":"3598333.88","SPT_WT_4h_0131119":"4685315.24"}],

"Metabolites":[{"Metabolite":"Cer d18:1/24:1","PubChem CID":"5283568"},{"Metabolite":"Cer d18:1/24:0","PubChem CID":"5283571"},{"Metabolite":"Cer d18:1/16:0","PubChem CID":"5283564"},{"Metabolite":"DHCer d18:0/16:0","PubChem CID":"5283572"},{"Metabolite":"DHCer d18:0/24:1","PubChem CID":"5283576"},{"Metabolite":"DHCer d18:0/24:0","PubChem CID":"5283577"}]
}

}