Summary of Study ST002274
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001455. The data can be accessed directly via it's Project DOI: 10.21228/M8NH7M This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST002274 |
| 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 |
| thekla.cordes@tu-bs.de | |
| Phone | 004953139155202 |
| Submit Date | 2022-08-31 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzXML |
| Analysis Type Detail | LC-MS |
| Release Date | 2022-09-16 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001455 |
| Project DOI: | doi: 10.21228/M8NH7M |
| 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) |
Subject:
| Subject ID: | SU002360 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Cell Biosource Or Supplier: | ATCC, CCL-247 |
| Cell Strain Details: | HCT116 |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Genotype | Treatment |
|---|---|---|---|
| SA217824 | SPT_C133W_15min_01311112 | C133W | 15min |
| SA217825 | SPT_C133W_15min_01311111 | C133W | 15min |
| SA217826 | SPT_C133W_15min_01311110 | C133W | 15min |
| SA217827 | SPT_C133W_1h_01311113 | C133W | 1h |
| SA217828 | SPT_C133W_1h_01311115 | C133W | 1h |
| SA217829 | SPT_C133W_1h_01311114 | C133W | 1h |
| SA217830 | SPT_C133W_4h_01311116 | C133W | 4h |
| SA217831 | SPT_C133W_4h_01311117 | C133W | 4h |
| SA217832 | SPT_C133W_4h_01311118 | C133W | 4h |
| SA217833 | SPT_WT_15min_0131112 | Wild-type | 15min |
| SA217834 | SPT_WT_15min_0131113 | Wild-type | 15min |
| SA217835 | SPT_WT_15min_0131111 | Wild-type | 15min |
| SA217836 | SPT_WT_1h_0131115 | Wild-type | 1h |
| SA217837 | SPT_WT_1h_0131114 | Wild-type | 1h |
| SA217838 | SPT_WT_1h_0131116 | Wild-type | 1h |
| SA217839 | SPT_WT_4h_0131117 | Wild-type | 4h |
| SA217840 | SPT_WT_4h_0131119 | Wild-type | 4h |
| SA217841 | SPT_WT_4h_0131118 | Wild-type | 4h |
| Showing results 1 to 18 of 18 |
Collection:
| Collection ID: | CO002353 |
| 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 ID: | TR002372 |
| Treatment Summary: | HCT116 expressing SPTLC1WT or SPTLC1C133W were cultured in media containing [U-13C16]palmitate for 15min, 1h, and 4h. |
Sample Preparation:
| Sampleprep ID: | SP002366 |
| 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. |
Combined analysis:
| Analysis ID | AN003716 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Vanquish |
| Column | Kinetex C8 (150 x 3mm,2.6um,100Å) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | POSITIVE |
| Units | ion counts |
Chromatography:
| Chromatography ID: | CH002753 |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Kinetex C8 (150 x 3mm,2.6um,100Å) |
| Column Temperature: | 40 |
| Flow Gradient: | 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. |
| Flow Rate: | 0.5 mL/min |
| Solvent A: | 100% water; 0.2% formic acid; 2 mM ammonium formate |
| Solvent B: | 100% methanol; 0.2% formic acid; 1 mM ammonium formate |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003465 |
| Analysis ID: | AN003716 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| 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. |
| Ion Mode: | POSITIVE |
