Summary of Study ST003531

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 PR002173. The data can be accessed directly via it's Project DOI: 10.21228/M8R23J This work is supported by NIH grant, U2C- DK119886.

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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.

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Study IDST003531
Study TitleMicropeptide hSPAR, a glutamine regulator, suppresses tumor growth via TRIM21-P27KIP1-mTOR pathway - human MDA-MB-231 breast cancer cell
Study SummaryhSPAR, a microprotein, is capable of specifically inhibiting the mTOR signaling activity and cell proliferation in MDA-MB-231 cells. Glutamine, an essential amino acid, plays a crucial role in regulating the mTOR signal. Our data indicate that in MDA-MB-231 cells, overexpressing hSPAR inhibits glutamine uptake, consequently suppressing the activation of the mTOR signaling.
Institute
University of Science and Technology of China
Last NameWang
First NameWei
AddressDivision of Life Sciences and Medicine, 443 Huangshan Road, Hefei city, Anhui Province
EmailWW571@mail.ustc.edu.cn
Phone18604523231
Submit Date2024-10-09
Raw Data AvailableYes
Raw Data File Type(s)mzML
Analysis Type DetailLC-MS
Release Date2025-01-06
Release Version1
Wei Wang Wei Wang
https://dx.doi.org/10.21228/M8R23J
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR002173
Project DOI:doi: 10.21228/M8R23J
Project Title:Micropeptide hSPAR, a glutamine regulator, suppresses tumor growth via TRIM21-P27KIP1-mTOR pathway
Project Summary:The mammalian target of rapamycin (mTOR) plays pivotal roles in cancer growth control upon amino acid response. Recently, cyclin-dependent kinase (CDK) inhibitor cyclin-dependent kinase inhibitor 1B (CDKN1B or P27KIP1) 1 has been reported as a non-canonical inhibitor to mTOR signaling in mouse embryo fibroblasts (MEFs). However, the mechanisms underlying P27KIP1-mTOR axis are yet-to-be uncovered. Here, we find that micropeptide human small regulatory polypeptide of amino acid response (hSPAR), through its C-terminus (hSPAR-C), inhibits E3 ligase tripartite motif containing 21 (TRIM21)-mediated P27KIP1 degradation and causes P27KIP1’s cytoplasmic accumulation in breast cancer cells. Interestingly, hSPAR/hSPAR-C also serves as an inhibitor to glutamine transporter SLC38A2 and remarkably decreases the cellular glutamine level specifically in cancer cells. The resulted glutamine deprivation sequentially triggers translocation of cytoplasmic P27KIP1 to lysosomes, where P27KIP1 disrupts Ragulator complex and suppresses mTOR complex 1 (mTORC1) assembly. Administration of hSPAR or hSPAR-C dramatically impedes breast cancer cell proliferation and xenografic tumor growth. Collectively, we define hSPAR as an intrinsic molecule to control cellular glutamine level and a previously-unidentified tumor suppressor by promoting accumulation and lysosomal-localization of P27KIP1 to inhibit mTORC1 assembly.
Institute:University of Science and Technology of China
Last Name:Wang
First Name:Wei
Address:Division of Life Sciences and Medicine, 443 Huangshan Road, Hefei city, Anhui Province, 230022, China
Email:WW571@mail.ustc.edu.cn
Phone:18604523231

Subject:

Subject ID:SU003660
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606
Species Group:Mammals

Factors:

Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Sample source Transfection
SA387354ATG4MDA-MB-231 breast cancer cell ATG
SA387355ATG5MDA-MB-231 breast cancer cell ATG
SA387356ATG6MDA-MB-231 breast cancer cell ATG
SA387357Ctrl4MDA-MB-231 breast cancer cell Ctrl
SA387358Ctrl5MDA-MB-231 breast cancer cell Ctrl
SA387359Ctrl6MDA-MB-231 breast cancer cell Ctrl
SA387360hsPAR4MDA-MB-231 breast cancer cell hsPAR
SA387361hsPAR5MDA-MB-231 breast cancer cell hsPAR
SA387362hsPAR6MDA-MB-231 breast cancer cell hsPAR
SA387363hsPAR_C4MDA-MB-231 breast cancer cell hsPAR_C
SA387364hsPAR_C5MDA-MB-231 breast cancer cell hsPAR_C
SA387365hsPAR_C6MDA-MB-231 breast cancer cell hsPAR_C
Showing results 1 to 12 of 12

Collection:

Collection ID:CO003653
Collection Summary:The MDA-MB-231 breast cancer cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), containing 10% fetal bovine serum and 1% penicillin-streptomycin under humidified atmosphere of 5% CO2 at 37°C. Ce11s were counted, washed with cold PBS and then flash-frozen in liquid N2.
Sample Type:Breast cancer cells

Treatment:

Treatment ID:TR003669
Treatment Summary:MDA-MB-231 breast cancer cells were transfected with empty vector, ΔATG1+2, hSPAR or hSPAR-C for 48 hours.

Sample Preparation:

Sampleprep ID:SP003667
Sampleprep Summary:The sample was thawed on ice, 100 μL of ultrapure water extract (containing protease inhibitors, PMSF and EDTA) was added to resuspend the cell pellet. Divide 50 μL cell suspension and add 200 µL of methanol (precooled at -20°C) and vortexed for 2 min under the condition of 2500 rpm. The sample was frozen in liquid nitrogen for 5 min, removed on ice for 5 min, after that, the sample was vortexed for 2 min.The previous step was repeated for 3 times. The sample was centrifuged at 12000 rpm for 10 min at 4°C. Take 200 μL of supernatant into a new centrifuge tube and place the supernatant in -20°C refrigerator for 30 min. Then the supernatant was centrifuged at 12000 rpm for 10 min at 4°C. After centrifugation, transfer 180 μL of supernatant through Protein Precipitation Plate for further LC-MS analysis. The remaining 50 μL cell suspension was frozen and thawed for 3 times, centrifuged at 12,000 rpm for 10 min, and the supernatant was taken to determine the protein concentration by BCA Protein Assay kit.
Sampleprep Protocol Filename:USTC_Protocol.pdf

Combined analysis:

Analysis ID AN005800 AN005801
Analysis type MS MS
Chromatography type HILIC HILIC
Chromatography system SCIEX ExionLC AD SCIEX ExionLC AD
Column Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um) Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um)
MS Type ESI ESI
MS instrument type QTRAP QTRAP
MS instrument name ABI Sciex 5500 QTrap ABI Sciex 5500 QTrap
Ion Mode POSITIVE NEGATIVE
Units ng/mg of cells ng/mg of cells

Chromatography:

Chromatography ID:CH004405
Methods Filename:USTC_Protocol.pdf
Instrument Name:SCIEX ExionLC AD
Column Name:Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um)
Column Temperature:40°C
Flow Gradient:The gradient was started at 90% B at 0-1.2 min, decreased to 60% B at 9 min, 40% B at 10-11 min, finaly ramped back to 90% B at 11.01-15 min
Flow Rate:0.4 mL/min
Solvent A:100% water; 2 mM ammonium acetate; 0.04% formic acid
Solvent B:100% acetonitrile; 2 mM ammonium acetate; 0.04% formic acid
Chromatography Type:HILIC

MS:

MS ID:MS005520
Analysis ID:AN005800
Instrument Name:ABI Sciex 5500 QTrap
Instrument Type:QTRAP
MS Type:ESI
MS Comments:-
Ion Mode:POSITIVE
Analysis Protocol File:USTC_Protocol.pdf
  
MS ID:MS005521
Analysis ID:AN005801
Instrument Name:ABI Sciex 5500 QTrap
Instrument Type:QTRAP
MS Type:ESI
MS Comments:-
Ion Mode:NEGATIVE
Analysis Protocol File:USTC_Protocol.pdf
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