#METABOLOMICS WORKBENCH SVM_20240430_040736 DATATRACK_ID:4802 STUDY_ID:ST003221 ANALYSIS_ID:AN005282 PROJECT_ID:PR002009
VERSION             	1
CREATED_ON             	May 24, 2024, 3:19 pm
#PROJECT
PR:PROJECT_TITLE                 	NMR-based lipidomics in Breast Cancer
PR:PROJECT_SUMMARY               	Recurrent and metastatic breast cancer is frequently treatment resistant. A
PR:PROJECT_SUMMARY               	wealth of evidence suggests that reprogrammed lipid metabolism supports cancer
PR:PROJECT_SUMMARY               	recurrence. Overexpression of the RON and DEK proteins in breast cancer is
PR:PROJECT_SUMMARY               	associated with poor outcome. Both proteins promote cancer metastasis in
PR:PROJECT_SUMMARY               	laboratory models, but effects on lipid metabolite levels remain unknown. To
PR:PROJECT_SUMMARY               	measure RON- and DEK-dependent steady-state lipid metabolite levels, an Nuclear
PR:PROJECT_SUMMARY               	Magnetic Resonance (NMR)-based approach was utilized. The observed differences
PR:PROJECT_SUMMARY               	were then used to identify a lipid metabolism-related gene expression signature
PR:PROJECT_SUMMARY               	that is prognostic of overall survival (OS), distant metastasis free survival
PR:PROJECT_SUMMARY               	(DMFS), post-progression survival (PPS), and recurrence free survival (RFS) in
PR:PROJECT_SUMMARY               	patients with breast cancer. RON loss led to decreased cholesterol and
PR:PROJECT_SUMMARY               	sphingomyelin levels, while DEK loss increased total fatty acid levels and
PR:PROJECT_SUMMARY               	decreased free glycerol levels. Lipid-related genes were then queried to define
PR:PROJECT_SUMMARY               	a signature that predicts breast, ovarian, and lung cancer patient survival.
PR:PROJECT_SUMMARY               	Taken together, RON and DEK differentially regulate lipid metabolism in a manner
PR:PROJECT_SUMMARY               	that predicts and may promote breast cancer metastasis and recurrence.
PR:INSTITUTE                     	Cincinnati Children's Hospital Medical Center
PR:LAST_NAME                     	Vicente-Munoz
PR:FIRST_NAME                    	Sara
PR:ADDRESS                       	3333 Burnet Ave, Cincinnati, OH, 45206, USA
PR:EMAIL                         	Sara.VicenteMunoz@cchmc.org
PR:PHONE                         	5135172083
#STUDY
ST:STUDY_TITLE                   	Untargeted NMR-lipidomic study in R7, R7sgRON, R7shDEK cells
ST:STUDY_SUMMARY                 	Recurrent and metastatic breast cancer is frequently treatment resistant. A
ST:STUDY_SUMMARY                 	wealth of evidence suggests that reprogrammed lipid metabolism supports cancer
ST:STUDY_SUMMARY                 	recurrence. Overexpression of the RON and DEK proteins in breast cancer is
ST:STUDY_SUMMARY                 	associated with poor outcome. Both proteins promote cancer metastasis in
ST:STUDY_SUMMARY                 	laboratory models, but effects on lipid metabolite levels remain unknown. To
ST:STUDY_SUMMARY                 	measure RON- and DEK-dependent steady-state lipid metabolite levels, an Nuclear
ST:STUDY_SUMMARY                 	Magnetic Resonance (NMR)-based approach was utilized. The observed differences
ST:STUDY_SUMMARY                 	were then used to identify a lipid metabolism-related gene expression signature
ST:STUDY_SUMMARY                 	that is prognostic of overall survival (OS), distant metastasis free survival
ST:STUDY_SUMMARY                 	(DMFS), post-progression survival (PPS), and recurrence free survival (RFS) in
ST:STUDY_SUMMARY                 	patients with breast cancer. RON loss led to decreased cholesterol and
ST:STUDY_SUMMARY                 	sphingomyelin levels, while DEK loss increased total fatty acid levels and
ST:STUDY_SUMMARY                 	decreased free glycerol levels. Lipid-related genes were then queried to define
ST:STUDY_SUMMARY                 	a signature that predicts breast, ovarian, and lung cancer patient survival.
ST:STUDY_SUMMARY                 	Taken together, RON and DEK differentially regulate lipid metabolism in a manner
ST:STUDY_SUMMARY                 	that predicts and may promote breast cancer metastasis and recurrence.
ST:INSTITUTE                     	Cincinnati Children's Hospital Medical Center
ST:LAST_NAME                     	Vicente-Munoz
ST:FIRST_NAME                    	Sara
ST:ADDRESS                       	3333 Burnet Ave
ST:EMAIL                         	Sara.VicenteMunoz@cchmc.org
ST:PHONE                         	5135172083
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Mus musculus
SU:TAXONOMY_ID                   	10090
#FACTORS
#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           	-	R7-1	Sample source:Murine breast cancer cells | Genotype:R7	RAW_FILE_NAME(Raw_file_data)=Wells_BC_SET1_3Cell-lines_LIPIDS_042522.1.fid
SUBJECT_SAMPLE_FACTORS           	-	R7-2	Sample source:Murine breast cancer cells | Genotype:R7	RAW_FILE_NAME(Raw_file_data)=Wells_BC_SET1_3Cell-lines_LIPIDS_042522.4.fid
SUBJECT_SAMPLE_FACTORS           	-	R7-3	Sample source:Murine breast cancer cells | Genotype:R7	RAW_FILE_NAME(Raw_file_data)=Wells_BC_SET1_3Cell-lines_LIPIDS_042522.7.fid
SUBJECT_SAMPLE_FACTORS           	-	R7-4	Sample source:Murine breast cancer cells | Genotype:R7	RAW_FILE_NAME(Raw_file_data)=Wells_BC_SET1_3Cell-lines_LIPIDS_042522.10.fid
SUBJECT_SAMPLE_FACTORS           	-	R7-5	Sample source:Murine breast cancer cells | Genotype:R7	RAW_FILE_NAME(Raw_file_data)=Wells_BC_SET1_3Cell-lines_LIPIDS_042522.13.fid
SUBJECT_SAMPLE_FACTORS           	-	R7-6	Sample source:Murine breast cancer cells | Genotype:R7	RAW_FILE_NAME(Raw_file_data)=Wells_BC_SET1_3Cell-lines_LIPIDS_042522.16.fid
SUBJECT_SAMPLE_FACTORS           	-	R7-7	Sample source:Murine breast cancer cells | Genotype:R7	RAW_FILE_NAME(Raw_file_data)=Wells_BC_SET1_3Cell-lines_LIPIDS_042522.19.fid
SUBJECT_SAMPLE_FACTORS           	-	RON-1	Sample source:Murine breast cancer cells | Genotype:R7sgRON	RAW_FILE_NAME(Raw_file_data)=Wells_BC_SET1_3Cell-lines_LIPIDS_042522.2.fid
SUBJECT_SAMPLE_FACTORS           	-	RON-2	Sample source:Murine breast cancer cells | Genotype:R7sgRON	RAW_FILE_NAME(Raw_file_data)=Wells_BC_SET1_3Cell-lines_LIPIDS_042522.5.fid
SUBJECT_SAMPLE_FACTORS           	-	RON-3	Sample source:Murine breast cancer cells | Genotype:R7sgRON	RAW_FILE_NAME(Raw_file_data)=Wells_BC_SET1_3Cell-lines_LIPIDS_042522.8.fid
SUBJECT_SAMPLE_FACTORS           	-	RON-4	Sample source:Murine breast cancer cells | Genotype:R7sgRON	RAW_FILE_NAME(Raw_file_data)=Wells_BC_SET1_3Cell-lines_LIPIDS_042522.11.fid
SUBJECT_SAMPLE_FACTORS           	-	RON-5	Sample source:Murine breast cancer cells | Genotype:R7sgRON	RAW_FILE_NAME(Raw_file_data)=Wells_BC_SET1_3Cell-lines_LIPIDS_042522.14.fid
SUBJECT_SAMPLE_FACTORS           	-	RON-6	Sample source:Murine breast cancer cells | Genotype:R7sgRON	RAW_FILE_NAME(Raw_file_data)=Wells_BC_SET1_3Cell-lines_LIPIDS_042522.17.fid
SUBJECT_SAMPLE_FACTORS           	-	RON-7	Sample source:Murine breast cancer cells | Genotype:R7sgRON	RAW_FILE_NAME(Raw_file_data)=Wells_BC_SET1_3Cell-lines_LIPIDS_042522.20.fid
SUBJECT_SAMPLE_FACTORS           	-	DEK-1	Sample source:Murine breast cancer cells | Genotype:R7shDEK	RAW_FILE_NAME(Raw_file_data)=Wells_BC_SET1_3Cell-lines_LIPIDS_042522.3.fid
SUBJECT_SAMPLE_FACTORS           	-	DEK-2	Sample source:Murine breast cancer cells | Genotype:R7shDEK	RAW_FILE_NAME(Raw_file_data)=Wells_BC_SET1_3Cell-lines_LIPIDS_042522.6.fid
SUBJECT_SAMPLE_FACTORS           	-	DEK-3	Sample source:Murine breast cancer cells | Genotype:R7shDEK	RAW_FILE_NAME(Raw_file_data)=Wells_BC_SET1_3Cell-lines_LIPIDS_042522.9.fid
SUBJECT_SAMPLE_FACTORS           	-	DEK-4	Sample source:Murine breast cancer cells | Genotype:R7shDEK	RAW_FILE_NAME(Raw_file_data)=Wells_BC_SET1_3Cell-lines_LIPIDS_042522.12.fid
SUBJECT_SAMPLE_FACTORS           	-	DEK-5	Sample source:Murine breast cancer cells | Genotype:R7shDEK	RAW_FILE_NAME(Raw_file_data)=Wells_BC_SET1_3Cell-lines_LIPIDS_042522.15.fid
SUBJECT_SAMPLE_FACTORS           	-	DEK-6	Sample source:Murine breast cancer cells | Genotype:R7shDEK	RAW_FILE_NAME(Raw_file_data)=Wells_BC_SET1_3Cell-lines_LIPIDS_042522.18.fid
SUBJECT_SAMPLE_FACTORS           	-	DEK-7	Sample source:Murine breast cancer cells | Genotype:R7shDEK	RAW_FILE_NAME(Raw_file_data)=Wells_BC_SET1_3Cell-lines_LIPIDS_042522.21.fid
#COLLECTION
CO:COLLECTION_SUMMARY            	Cells were washed three times with ice-cold phosphate-buffered saline, followed
CO:COLLECTION_SUMMARY            	by subsequent quenching with ice cold acetonitrile (CH3CN)and the addition of
CO:COLLECTION_SUMMARY            	nano-pure water (CH3CN/H2O at 2:1.5 (V/V)) to facilitate cell scraping and
CO:COLLECTION_SUMMARY            	collection.
CO:COLLECTION_PROTOCOL_FILENAME  	BC-SET1-MM.pdf
CO:SAMPLE_TYPE                   	Breast cancer cells
#TREATMENT
TR:TREATMENT_SUMMARY             	R7 (control), R7sgRON (RON targeted), R7shDEK (DEK targeted) murine breast
TR:TREATMENT_SUMMARY             	cancer cell lines were cultured in complete Dulbecco’s Modified Eagle Medium
TR:TREATMENT_SUMMARY             	(DMEM) containing 5% FBS, 1% penicillin-streptomycin, and 0.2% fungizone.
TR:TREATMENT_SUMMARY             	R7shDEK were maintained with 1 μg/mL of puromycin for selection. For NMR
TR:TREATMENT_SUMMARY             	experiments, cells were seeded in 10 cm-plates in complete DMEM containing 5%
TR:TREATMENT_SUMMARY             	dialyzed FBS.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Dried organic phases (lipids) were reconstituted in 220 µL of 100% methanol-d4
SP:SAMPLEPREP_SUMMARY            	containing 0.05% v/v of tetramethylsilane (TMS) (Cambridge Isotopes Lab.
SP:SAMPLEPREP_SUMMARY            	Andover, MA), vortexed and centrifuged at room temperature. 200 µL of the
SP:SAMPLEPREP_SUMMARY            	supernatant were transferred into 3 mm NMR tube.
SP:PROCESSING_STORAGE_CONDITIONS 	On ice
SP:EXTRACT_STORAGE               	On ice
#ANALYSIS
AN:ANALYSIS_PROTOCOL_FILE        	BC-SET1-MM.pdf
#NMR
NM:INSTRUMENT_NAME               	Bruker Avance III HD 600 MHz spectrometer
NM:INSTRUMENT_TYPE               	FT-NMR
NM:NMR_EXPERIMENT_TYPE           	1D-1H
NM:SPECTROMETER_FREQUENCY        	600 MHz
NM:NMR_PROBE                     	5 mm Broad Band Observed (BBO) Prodigy probe
NM:NMR_SOLVENT                   	MeOD
NM:NMR_TUBE_SIZE                 	3 mm
NM:SHIMMING_METHOD               	Topshim
NM:PULSE_SEQUENCE                	noesygppr1d
NM:TEMPERATURE                   	15
#NMR_METABOLITE_DATA
NMR_METABOLITE_DATA:UNITS	relative abundance
NMR_METABOLITE_DATA_START
Samples	R7-1	R7-2	R7-3	R7-4	R7-5	R7-6	R7-7	RON-1	RON-2	RON-3	RON-4	RON-5	RON-6	RON-7	DEK-1	DEK-2	DEK-3	DEK-4	DEK-5	DEK-6	DEK-7
Factors	Sample source:Murine breast cancer cells | Genotype:R7	Sample source:Murine breast cancer cells | Genotype:R7	Sample source:Murine breast cancer cells | Genotype:R7	Sample source:Murine breast cancer cells | Genotype:R7	Sample source:Murine breast cancer cells | Genotype:R7	Sample source:Murine breast cancer cells | Genotype:R7	Sample source:Murine breast cancer cells | Genotype:R7	Sample source:Murine breast cancer cells | Genotype:R7sgRON	Sample source:Murine breast cancer cells | Genotype:R7sgRON	Sample source:Murine breast cancer cells | Genotype:R7sgRON	Sample source:Murine breast cancer cells | Genotype:R7sgRON	Sample source:Murine breast cancer cells | Genotype:R7sgRON	Sample source:Murine breast cancer cells | Genotype:R7sgRON	Sample source:Murine breast cancer cells | Genotype:R7sgRON	Sample source:Murine breast cancer cells | Genotype:R7shDEK	Sample source:Murine breast cancer cells | Genotype:R7shDEK	Sample source:Murine breast cancer cells | Genotype:R7shDEK	Sample source:Murine breast cancer cells | Genotype:R7shDEK	Sample source:Murine breast cancer cells | Genotype:R7shDEK	Sample source:Murine breast cancer cells | Genotype:R7shDEK	Sample source:Murine breast cancer cells | Genotype:R7shDEK
C, CE18-CH3	1.019785	0.991714	0.936229	0.942712	1.023911	0.981517	1.104131	0.918789	0.888025	0.861944	0.868692	0.898992	0.889757	0.939382	1.025636	1.026795	1.002991	1.011833	0.994391	1.160186	1.105826
C, CE26-CH3, 27-CH3	1.019028	1.004171	0.936884	0.939011	1.006318	0.992724	1.101864	0.866441	0.902419	0.836737	0.877214	0.902482	0.888166	0.925630	1.014697	1.048286	1.033791	1.047226	1.131149	1.202605	1.083340
FA-chain-(methyls)	0.962907	1.012673	0.933818	0.953323	1.104314	1.144789	0.888176	1.124852	1.824028	1.107799	0.971171	1.142930	1.095645	0.977292	1.021141	1.112884	0.976507	1.119565	1.037078	1.010435	0.971011
C, CE 21-CH3, 9-CH	1.124659	1.040626	0.963198	0.931559	1.067422	0.966554	0.905982	0.870504	0.835207	0.837651	0.856248	0.977352	0.825946	0.924004	1.054810	1.058860	1.017248	1.056493	1.250278	1.335771	0.577510
C, CE 19-CH3	0.953142	0.999645	0.945764	0.949910	1.007551	1.018022	1.125965	0.951469	0.940020	0.836646	0.903862	0.903249	0.891834	1.003472	0.918336	0.934838	0.940755	0.911035	0.969728	1.103723	1.062922
FA-chain-(methylenes)	1.065682	1.177443	0.886454	0.864555	0.898487	1.113036	0.994342	0.883190	0.954174	0.935843	0.932232	1.086324	2.635516	1.206516	0.934787	0.978963	1.121085	0.944886	0.965913	1.100694	1.307855
FA-chain-(β-methylenes)	0.866653	1.037480	0.937959	1.068419	0.901169	1.119158	1.069163	0.970952	0.903256	1.003328	0.896619	0.980632	0.931279	1.055526	1.046438	0.926419	0.895353	0.993844	0.896095	0.973364	1.159829
FA-chain-(mono-allylic CH)	0.171566	0.354401	0.168334	0.179743	0.191635	0.173649	5.760673	0.178225	0.161736	0.157926	0.176163	0.153785	0.181169	0.144719	0.189631	0.169612	0.181977	0.177095	0.168218	0.182320	0.177270
Linoleic acid (18:2)	0.342176	0.562110	0.381710	0.403528	0.431019	0.432620	4.446837	0.338801	0.359420	0.465082	0.393222	0.386267	0.426908	0.352449	0.412327	0.579594	0.440851	0.430836	0.600788	0.640564	0.468236
FA-chain-(α-CH2)	1.026755	0.994753	0.973677	0.984714	1.018915	0.990973	1.010212	1.031753	1.056809	1.060049	1.043645	1.048872	1.034095	0.989592	1.248701	1.081665	1.119289	1.178196	1.084494	1.017675	1.168404
FA-chain-(α-methylenes)	1.017250	0.984392	0.986966	1.050629	1.055612	1.044800	0.860351	1.058696	1.047661	1.190761	0.996957	0.990438	0.970455	0.920754	1.235179	1.034847	1.068965	1.101110	1.056543	0.865743	0.987731
FA-chain-(di-allylic -methylenes)-linoleic acid (18:2)	1.017894	1.076923	0.894840	0.951600	0.950176	1.071693	1.036874	0.884119	1.072958	0.862960	0.947348	1.024402	0.752509	0.662613	1.250300	1.309529	1.373848	1.148100	1.113705	1.053476	0.948254
PUFA (di-allylic -methylenes)	0.856618	1.083708	1.004830	0.923079	1.041327	1.061893	1.028545	0.944379	0.981685	0.956244	1.010748	0.984365	0.968886	0.879057	0.871884	0.916592	1.070021	0.910592	0.930244	0.985212	1.042125
GPE, PL x-CH2-CH2-NH2	0.946863	0.869849	0.981385	1.237124	0.923286	0.916660	1.124833	0.996690	0.980175	0.848210	0.926082	1.022463	0.827428	0.677089	1.018070	0.992202	1.106472	0.986538	0.824605	1.101310	0.747995
Free glycerol C1 3H	0.947965	0.994811	0.902745	0.933806	1.066300	0.988440	1.165933	1.004860	0.970310	1.074603	1.206192	1.268579	1.038539	1.121442	0.680762	0.638088	0.933697	0.686522	0.890486	0.745131	0.846497
PC - OCH2-CH2-N+	0.999919	0.998443	0.984642	0.983886	0.964731	1.018571	1.049808	1.008524	0.991551	0.997410	0.990835	1.052364	0.994977	0.978305	1.006610	1.000548	1.016158	0.973843	0.932691	1.048806	1.070755
Free glycerol C2 H	1.003998	0.935237	1.023992	0.966739	1.049556	0.972560	1.047917	1.066769	0.953541	1.044684	1.258610	1.212588	1.024017	1.094670	0.716692	0.690903	0.884375	0.703419	0.862723	0.740233	0.876100
Glyceril C3H	1.058675	0.967750	0.975766	0.986671	1.029637	1.024311	0.957189	0.985165	1.012845	0.928426	1.001247	0.968718	0.945983	0.921314	1.033415	1.095968	1.087944	1.065527	1.054566	1.058544	1.076710
Triglycerides CH2OR1 - CHOR2 - CH2OR3_A	0.982281	0.987643	0.964329	1.011377	1.008934	0.971262	1.074175	0.962749	0.993052	0.980256	0.987666	1.005387	0.996703	0.966958	1.101250	1.121039	1.102252	0.298050	1.110512	1.056259	1.071852
Phosphatidylcholine -CH2-CH2-N+	0.970315	0.977679	0.992063	1.047651	1.028217	1.062564	0.921511	1.024635	0.920025	0.958931	1.027353	0.957483	0.998465	0.965692	1.011147	1.018725	1.042784	1.027591	1.024880	1.063642	1.095751
Triglycerides CH2OR1 - CHOR2 - CH2OR3_B	0.949273	1.003437	1.011091	0.987229	1.000181	1.065610	0.983180	0.974000	0.959691	0.970952	1.024255	0.981024	0.982331	0.930394	1.081409	1.093893	1.105088	1.067869	1.080877	1.111000	1.110077
FA chain (olefinic)	0.848019	1.090073	0.997780	0.984813	1.022525	1.044714	1.012077	0.949540	0.980611	0.943838	0.943693	1.066669	0.967116	0.974484	1.016986	1.074591	1.107598	1.016557	1.031616	1.110666	1.193621
Plasmalogen (olefinic)	0.971515	0.912784	0.859186	0.992241	0.893221	0.964093	1.406959	1.069633	1.110275	1.066622	0.971581	1.506181	1.011220	0.974853	0.911567	0.979167	1.330806	0.924072	0.847954	0.891232	0.875286
Phenolic glycolipids -CH-(ring)	0.932502	1.300065	0.542218	1.047650	1.600529	1.267119	0.309916	1.482822	1.398562	1.546243	1.105671	2.364096	1.748299	1.109540	0.628753	0.642002	1.105608	0.695670	1.194179	1.551172	0.906355
Sphingomyelin (olefinic)	0.959986	0.923020	1.003310	1.119274	0.809830	0.876547	1.308033	0.784593	0.825130	0.823811	0.679888	0.961253	0.734670	0.549097	0.904839	1.045472	0.759057	0.982365	0.776063	0.747999	0.914867
NMR_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name
C, CE18-CH3
C, CE26-CH3, 27-CH3
FA-chain-(methyls)
C, CE 21-CH3, 9-CH
C, CE 19-CH3
FA-chain-(methylenes)
FA-chain-(β-methylenes)
FA-chain-(mono-allylic CH)
Linoleic acid (18:2)
FA-chain-(α-CH2)
FA-chain-(α-methylenes)
FA-chain-(di-allylic -methylenes)-linoleic acid (18:2)
PUFA (di-allylic -methylenes)
GPE, PL x-CH2-CH2-NH2
Free glycerol C1 3H
PC - OCH2-CH2-N+
Free glycerol C2 H
Glyceril C3H
Triglycerides CH2OR1 - CHOR2 - CH2OR3_A
Phosphatidylcholine -CH2-CH2-N+
Triglycerides CH2OR1 - CHOR2 - CH2OR3_B
FA chain (olefinic)
Plasmalogen (olefinic)
Phenolic glycolipids -CH-(ring)
Sphingomyelin (olefinic)
METABOLITES_END
#END