#METABOLOMICS WORKBENCH tcordes_20220831_013111 DATATRACK_ID:3441 STUDY_ID:ST002274 ANALYSIS_ID:AN003716 PROJECT_ID:PR001455 VERSION 1 CREATED_ON September 1, 2022, 10:56 am #PROJECT PR:PROJECT_TITLE 1-deoxysphingolipid synthesis compromises anchorage-independent growth and PR:PROJECT_TITLE plasma membrane endocytosis in cancer cells PR:PROJECT_TYPE Manuscript PR:PROJECT_SUMMARY Serine palmitoyltransferase (SPT) predominantly incorporates serine and fatty PR:PROJECT_SUMMARY acyl-CoAs into diverse sphingolipids that serve as structural components of PR:PROJECT_SUMMARY membranes and signaling molecules within or amongst cells. However, SPT also PR:PROJECT_SUMMARY uses alanine as a substrate in the contexts of low serine availability, alanine PR:PROJECT_SUMMARY accumulation, or disease-causing mutations in hereditary sensory neuropathy type PR:PROJECT_SUMMARY I (HSAN1), resulting in the synthesis and accumulation of 1-deoxysphingolipids. PR:PROJECT_SUMMARY These species promote cytotoxicity in neurons and impact diverse cellular PR:PROJECT_SUMMARY phenotypes, including suppression of anchorage-independent cancer cell growth. PR:PROJECT_SUMMARY While altered serine and alanine can promote 1-deoxysphingolipid synthesis, they PR:PROJECT_SUMMARY impact numerous other metabolic pathways important for cancer cells. Here we PR:PROJECT_SUMMARY combined isotope tracing, quantitative metabolomics, and functional studies to PR:PROJECT_SUMMARY better understand the mechanistic drivers of 1-deoxysphingolipid toxicity in PR:PROJECT_SUMMARY cancer cells. Both alanine treatment and SPTLC1C133W expression induce PR:PROJECT_SUMMARY 1-deoxy(dihydro)ceramide synthesis and accumulation but fail to broadly impact PR:PROJECT_SUMMARY intermediary metabolism, abundances of other lipids, or growth of adherent PR:PROJECT_SUMMARY cells. However, spheroid culture and soft agar colony formation were compromised PR:PROJECT_SUMMARY when endogenous 1-deoxysphingolipid synthesis was induced via SPTLC1C133W PR:PROJECT_SUMMARY expression. Consistent with these impacts on anchorage-independent cell growth, PR:PROJECT_SUMMARY we observed that 1-deoxysphingolipid synthesis reduced plasma membrane PR:PROJECT_SUMMARY endocytosis. These results highlight a potential role for SPT promiscuity in PR:PROJECT_SUMMARY linking altered amino acid metabolism to plasma membrane endocytosis. PR:INSTITUTE TU Braunschweig PR:LAST_NAME Cordes PR:FIRST_NAME Thekla PR:ADDRESS Rebenring 56, Braunschweig, Lower Saxony, 38106, Germany PR:EMAIL thekla.cordes@tu-bs.de PR:PHONE 04953139155202 PR:FUNDING_SOURCE NIH NCI R01CA234245 and R50CA252146 PR:PUBLICATIONS JLR accepted PR:CONTRIBUTORS Thekla Cordes (University of California San Diego; Salk Institute for Biological PR:CONTRIBUTORS Studies; TU Braunschweig), Christian Metallo (University of California, San PR:CONTRIBUTORS Diego; Salk Institute for Biological Studies) #STUDY ST:STUDY_TITLE 1-deoxysphingolipid synthesis compromises anchorage-independent growth and ST:STUDY_TITLE plasma membrane endocytosis in cancer cells ST:STUDY_SUMMARY Serine palmitoyltransferase (SPT) predominantly incorporates serine and fatty ST:STUDY_SUMMARY acyl-CoAs into diverse sphingolipids that serve as structural components of ST:STUDY_SUMMARY membranes and signaling molecules within or amongst cells. However, SPT also ST:STUDY_SUMMARY uses alanine as a substrate in the contexts of low serine availability, alanine ST:STUDY_SUMMARY accumulation, or diseasecausing mutations in hereditary sensory neuropathy type ST:STUDY_SUMMARY I (HSAN1), resulting in the synthesis and accumulation of 1-deoxysphingolipids. ST:STUDY_SUMMARY These species promote cytotoxicity in neurons and impact diverse cellular ST:STUDY_SUMMARY phenotypes, including suppression of anchorage-independent cancer cell growth. ST:STUDY_SUMMARY While altered serine and alanine can promote 1-deoxysphingolipid synthesis, they ST:STUDY_SUMMARY impact numerous other metabolic pathways important for cancer cells. Here we ST:STUDY_SUMMARY combined isotope tracing, quantitative metabolomics, and functional studies to ST:STUDY_SUMMARY better understand the mechanistic drivers of 1- deoxysphingolipid toxicity in ST:STUDY_SUMMARY cancer cells. Both alanine treatment and SPTLC1 C133W expression induce ST:STUDY_SUMMARY 1-deoxy(dihydro)ceramide synthesis and accumulation but fail to broadly impact ST:STUDY_SUMMARY intermediary metabolism, abundances of other lipids, or growth of adherent ST:STUDY_SUMMARY cells. However, spheroid culture and soft agar colony formation were compromised ST:STUDY_SUMMARY when endogenous 1-deoxysphingolipid synthesis was induced via SPTLC1 C133W ST:STUDY_SUMMARY expression. Consistent with these impacts on anchorageindependent cell growth, ST:STUDY_SUMMARY we observed that 1-deoxysphingolipid synthesis reduced plasma membrane ST:STUDY_SUMMARY endocytosis. These results highlight a potential role for SPT promiscuity in ST:STUDY_SUMMARY linking altered amino acid metabolism to plasma membrane endocytosis. ST:INSTITUTE Salk Institute for Biological Studies ST:LABORATORY Molecular and Cell Biology Laboratory (Christian Metallo) ST:LAST_NAME Cordes ST:FIRST_NAME Thekla ST:ADDRESS 10010 N Torrey Pines Rd, La Jolla, CA 92037, United States ST:EMAIL thekla.cordes@tu-bs.de ST:PHONE 004953139155202 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:CELL_BIOSOURCE_OR_SUPPLIER ATCC, CCL-247 SU:CELL_STRAIN_DETAILS HCT116 #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 - SPT_WT_15min_0131111 Genotype:Wild-type | Treatment:15min RAW_FILE_NAME=SPT_WT_15min_0131111.mzXML SUBJECT_SAMPLE_FACTORS - SPT_WT_15min_0131112 Genotype:Wild-type | Treatment:15min RAW_FILE_NAME=SPT_WT_15min_0131112.mzXML SUBJECT_SAMPLE_FACTORS - SPT_WT_15min_0131113 Genotype:Wild-type | Treatment:15min RAW_FILE_NAME=SPT_WT_15min_0131113.mzXML SUBJECT_SAMPLE_FACTORS - SPT_WT_1h_0131114 Genotype:Wild-type | Treatment:1h RAW_FILE_NAME=SPT_WT_1h_0131114.mzXML SUBJECT_SAMPLE_FACTORS - SPT_WT_1h_0131115 Genotype:Wild-type | Treatment:1h RAW_FILE_NAME=SPT_WT_1h_0131115.mzXML SUBJECT_SAMPLE_FACTORS - SPT_WT_1h_0131116 Genotype:Wild-type | Treatment:1h RAW_FILE_NAME=SPT_WT_1h_0131116.mzXML SUBJECT_SAMPLE_FACTORS - SPT_WT_4h_0131117 Genotype:Wild-type | Treatment:4h RAW_FILE_NAME=SPT_WT_4h_0131117.mzXML SUBJECT_SAMPLE_FACTORS - SPT_WT_4h_0131118 Genotype:Wild-type | Treatment:4h RAW_FILE_NAME=SPT_WT_4h_0131118.mzXML SUBJECT_SAMPLE_FACTORS - SPT_WT_4h_0131119 Genotype:Wild-type | Treatment:4h RAW_FILE_NAME=SPT_WT_4h_0131119.mzXML SUBJECT_SAMPLE_FACTORS - SPT_C133W_15min_01311110 Genotype:C133W | Treatment:15min RAW_FILE_NAME=SPT_C133W_15min_01311110.mzXML SUBJECT_SAMPLE_FACTORS - SPT_C133W_15min_01311111 Genotype:C133W | Treatment:15min RAW_FILE_NAME=SPT_C133W_15min_01311111.mzXML SUBJECT_SAMPLE_FACTORS - SPT_C133W_15min_01311112 Genotype:C133W | Treatment:15min RAW_FILE_NAME=SPT_C133W_15min_01311112.mzXML SUBJECT_SAMPLE_FACTORS - SPT_C133W_1h_01311113 Genotype:C133W | Treatment:1h RAW_FILE_NAME=SPT_C133W_1h_01311113.mzXML SUBJECT_SAMPLE_FACTORS - SPT_C133W_1h_01311114 Genotype:C133W | Treatment:1h RAW_FILE_NAME=SPT_C133W_1h_01311114.mzXML SUBJECT_SAMPLE_FACTORS - SPT_C133W_1h_01311115 Genotype:C133W | Treatment:1h RAW_FILE_NAME=SPT_C133W_1h_01311115.mzXML SUBJECT_SAMPLE_FACTORS - SPT_C133W_4h_01311116 Genotype:C133W | Treatment:4h RAW_FILE_NAME=SPT_C133W_4h_01311116.mzXML SUBJECT_SAMPLE_FACTORS - SPT_C133W_4h_01311117 Genotype:C133W | Treatment:4h RAW_FILE_NAME=SPT_C133W_4h_01311117.mzXML SUBJECT_SAMPLE_FACTORS - SPT_C133W_4h_01311118 Genotype:C133W | Treatment:4h RAW_FILE_NAME=SPT_C133W_4h_01311118.mzXML #COLLECTION CO:COLLECTION_SUMMARY For tracing studies with [U-13C16]palmitate, HCT116 cells were cultured in CO:COLLECTION_SUMMARY growth medium in the presence of 0.1 μg/ml doxycycline for 7 days before tracer CO:COLLECTION_SUMMARY start. Growth medium was replaced to DMEM medium containing 1% (v/v) delipidated CO:COLLECTION_SUMMARY FBS 24h prior tracer start and medium exchange again 1h prior tracer trace. CO:COLLECTION_SUMMARY [U-13C16]palmitate was noncovalently bound to fatty acid-free BSA and added to CO:COLLECTION_SUMMARY culture medium at 5% of the final volume (50 μM final concentration). Media was CO:COLLECTION_SUMMARY prewarmed to 37oC in a cell incubator with 5% CO2 and cells were traced for 15 CO:COLLECTION_SUMMARY min, 1 h, and 4 h. For targeted sphingolipid analysis, cells were washed with CO:COLLECTION_SUMMARY 0.9% (w/v) NaCl and extracted with 0.25 mL of −20°C methanol, 0.25 mL -20°C CO:COLLECTION_SUMMARY chloroform, and 0.1 mL of water. The tubes were vortexed for 5 min, centrifuged CO:COLLECTION_SUMMARY at 20,000 x g at 4°C for 5 min, and the lower organic phase was collected. The CO:COLLECTION_SUMMARY remaining polar phase was extracted with 2 μL formic acid and 0.25 mL of -20 CO:COLLECTION_SUMMARY °C chloroform. The organic phases were combined, dried under air, resuspended CO:COLLECTION_SUMMARY in 80 μL Buffer B (0.2% formic acid and 1 mM ammonium formate in methanol), CO:COLLECTION_SUMMARY sonicated for 10 min, and centrifuged for 10 min at 20,000 x g at 4 °C. The CO:COLLECTION_SUMMARY supernatant was transferred to LC vials containing glass inserts for analysis on CO:COLLECTION_SUMMARY a Q-Exactive LC-MS system. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY HCT116 expressing SPTLC1WT or SPTLC1C133W were cultured in media containing TR:TREATMENT_SUMMARY [U-13C16]palmitate for 15min, 1h, and 4h. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The samples were reconstituted in 80 μL Buffer B (0.2% formic acid and 1 mM SP:SAMPLEPREP_SUMMARY ammonium formate in methanol), sonicated for 10 min, and centrifuged for 10 min SP:SAMPLEPREP_SUMMARY at 20,000 x g at 4 °C. The supernatant was transferred to LC vials containing SP:SAMPLEPREP_SUMMARY glass inserts for analysis on a Q-Exactive LC-MS system. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Kinetex 2.6 μM C8 100 Å 150 x 3 mm LC column (Phenomenex) #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Christian Metallo #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS To quantify labeling on SL and deoxySL species from [U-13C16]palmitate a Q MS:MS_COMMENTS Exactive orbitrap mass spectrometer with a Vanquish Flex Binary UHPLC system MS:MS_COMMENTS (Thermo Scientific) was used with a Kinetex 2.6 μM C8 100 Å 150 x 3 mm LC MS:MS_COMMENTS column (Phenomenex) at 40°C. 5 μL of sample was injected. Chromatography was MS:MS_COMMENTS performed using a gradient of 2 mM ammonium formate and 0.2 % formic acid MS:MS_COMMENTS (mobile phase A) and 1 mM ammonium formate and 0.2 % formic acid in methanol MS:MS_COMMENTS (mobile phase B), at a flow rate of 0.5 mL/min. The LC gradient held at 82% B MS:MS_COMMENTS for 0-3 min, then ran from 82%-90% B in 3-4 min, then 90-99% in 4-18 min, held MS:MS_COMMENTS at 99% B for 7 min, then reduced from 99%-82% from 25-27 min, then held at 82% MS:MS_COMMENTS for a further 13 mins. Lipids were analyzed in positive mode using spray voltage MS:MS_COMMENTS 3 kV. Sweep gas flow was 5 arbitrary units, auxiliary gas flow 7 arbitrary units MS:MS_COMMENTS and sheath gas flow 50 arbitrary units, with a capillary temperature of 300°C. MS:MS_COMMENTS Full MS (scan range 150-2000 m/z) was used at 70 000 resolution with 1e6 MS:MS_COMMENTS automatic gain control and a maximum injection time of 200 ms. Data dependent MS:MS_COMMENTS MS2 (Top 6) mode at 17 500 resolution with automatic gain control set at 1e5 MS:MS_COMMENTS with a maximum injection time of 50 ms was used for peak identification, MS:MS_COMMENTS combined with known standards where possible. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS ion counts MS_METABOLITE_DATA_START Samples SPT_C133W_15min_01311110 SPT_C133W_15min_01311111 SPT_C133W_15min_01311112 SPT_C133W_1h_01311113 SPT_C133W_1h_01311114 SPT_C133W_1h_01311115 SPT_C133W_4h_01311116 SPT_C133W_4h_01311117 SPT_C133W_4h_01311118 SPT_WT_15min_0131111 SPT_WT_15min_0131112 SPT_WT_15min_0131113 SPT_WT_1h_0131114 SPT_WT_1h_0131115 SPT_WT_1h_0131116 SPT_WT_4h_0131117 SPT_WT_4h_0131118 SPT_WT_4h_0131119 Factors Genotype:C133W | Treatment:15min Genotype:C133W | Treatment:15min Genotype:C133W | Treatment:15min Genotype:C133W | Treatment:1h Genotype:C133W | Treatment:1h Genotype:C133W | Treatment:1h Genotype:C133W | Treatment:4h Genotype:C133W | Treatment:4h Genotype:C133W | Treatment:4h Genotype:Wild-type | Treatment:15min Genotype:Wild-type | Treatment:15min Genotype:Wild-type | Treatment:15min Genotype:Wild-type | Treatment:1h Genotype:Wild-type | Treatment:1h Genotype:Wild-type | Treatment:1h Genotype:Wild-type | Treatment:4h Genotype:Wild-type | Treatment:4h Genotype:Wild-type | Treatment:4h Cer d18:1/24:1 12151155.54 10205801.85 8119015.47 8740968.8 10892397.36 15546699.37 12183738.41 12657917.77 18556726.2 6894899.97 9097543.84 9707655.69 17412002.16 12209404.72 13858027.47 12063002.8 11806994.75 16937500.01 Cer d18:1/24:0 7101466.47 5577524.07 4432532.4 4698830.23 6376552.03 14126756 6879697.68 7353515.96 10070106.88 3853669.42 5582265.38 5752534.56 10445284.89 7196665.85 7380665.68 7220515.56 6381753.28 9152040.07 Cer d18:1/16:0 6830905.75 5798724.12 4213409.12 7502301.32 8367183.07 15262343.15 10244717.43 12104078.65 15641062.11 3568996.72 5195217.75 6178559.37 15647106.76 11124028.25 11924427.94 11680062.15 10332946.87 15824413.31 DHCer d18:0/16:0 22143391.7 13683684.41 11902550.8 25521948.28 42804508.34 20244795.8 14332535 14668814.13 27568758.85 12509405.84 15779144.91 19049310.39 83038917.05 54616598.64 66879766.37 8643847.96 7897394.81 24867557.38 DHCer d18:0/24:1 10448464.91 9268438.72 7853127.85 10089404.89 13007588.25 16109414.77 8624040.76 9767746.02 13622650.75 5822165.67 8011510.11 10128684.46 24503447.77 15607295.47 21391100.35 7333889.78 8990329.22 11652897.29 DHCer d18:0/24:0 3709621.98 3395960.05 2847665.38 3299140.61 4752275.78 11464046.7 3636223.26 4243709.45 5891759.15 2348186.72 2633300.84 3691289.04 8232248.05 5514211.93 7122051.07 3609123.2 3598333.88 4685315.24 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name PubChem CID Cer d18:1/24:1 5283568 Cer d18:1/24:0 5283571 Cer d18:1/16:0 5283564 DHCer d18:0/16:0 5283572 DHCer d18:0/24:1 5283576 DHCer d18:0/24:0 5283577 METABOLITES_END #END