#METABOLOMICS WORKBENCH rushingb_20240617_083339 DATATRACK_ID:4934 STUDY_ID:ST003263 ANALYSIS_ID:AN005347 PROJECT_ID:PR002025 VERSION 1 CREATED_ON 06-17-2024 #PROJECT PR:PROJECT_TITLE MAPK14/p38 Shapes the Molecular Landscape of Endometrial Cancer and Confers PR:PROJECT_TITLE Tumorigenic Characteristics PR:PROJECT_SUMMARY The molecular underpinnings of High Grade Endometrial Carcinoma (HGEC) PR:PROJECT_SUMMARY metastatic growth and survival are poorly understood and this knowledge gap is a PR:PROJECT_SUMMARY barrier to improving patient outcomes. Here we show that ascites-derived and PR:PROJECT_SUMMARY primary tumor HGEC cell lines in 3D spheroid culture (which faithfully PR:PROJECT_SUMMARY recapitulate key features of malignant peritoneal effusion) exhibit PR:PROJECT_SUMMARY fundamentally distinct transcriptomic, proteomic and metabolomic landscapes when PR:PROJECT_SUMMARY compared with conventional 2D monolayers. Using a new genetic screening platform PR:PROJECT_SUMMARY we identify MAPK14 (which encodes the protein kinase p38alpha) as a specific PR:PROJECT_SUMMARY requirement for HGEC growing in spheroid culture. We show that MAPK14/p38alpha PR:PROJECT_SUMMARY has broad roles in programing the phosphoproteome, transcriptome and metabolome PR:PROJECT_SUMMARY of HGEC spheroids, yet has negligible impact on monolayer cultures. Using PR:PROJECT_SUMMARY single-cell analysis, we show that MAPK14 is specifically required to sustain a PR:PROJECT_SUMMARY sub-population of spheroid cells that is enriched in cancer stemness markers. PR:PROJECT_SUMMARY MAPK14 also promotes cancer HGEC tumorigenicity in vivo. We conclude that PR:PROJECT_SUMMARY spheroid growth of HGEC activates unique biological programs, including p38alpha PR:PROJECT_SUMMARY signaling, that are highly relevant to malignant disease pathology and cannot be PR:PROJECT_SUMMARY captured using 2D culture models. Our work reveals MAPK14/p38alpha as a new PR:PROJECT_SUMMARY dependency and potential therapeutic target in HGEC. The experimental models and PR:PROJECT_SUMMARY comprehensive data sets presented here are a resource that will help provide a PR:PROJECT_SUMMARY better mechanistic understanding of HGEC biology and facilitate translational PR:PROJECT_SUMMARY advances. PR:INSTITUTE University of North Carolina at Chapel Hill PR:LAST_NAME Rushing PR:FIRST_NAME Blake PR:ADDRESS 500 Laureate Way, Kannapolis, NC, 28081, USA PR:EMAIL blake_rushing@unc.edu PR:PHONE +1 (704) 250-5000 PR:DOI http://dx.doi.org/10.21228/M8VJ86 #STUDY ST:STUDY_TITLE MAPK14/p38 Shapes the Molecular Landscape of Endometrial Cancer and Confers ST:STUDY_TITLE Tumorigenic Characteristics ST:STUDY_SUMMARY The molecular underpinnings of High Grade Endometrial Carcinoma (HGEC) ST:STUDY_SUMMARY metastatic growth and survival are poorly understood and this knowledge gap is a ST:STUDY_SUMMARY barrier to improving patient outcomes. Here we show that ascites-derived and ST:STUDY_SUMMARY primary tumor HGEC cell lines in 3D spheroid culture (which faithfully ST:STUDY_SUMMARY recapitulate key features of malignant peritoneal effusion) exhibit ST:STUDY_SUMMARY fundamentally distinct transcriptomic, proteomic and metabolomic landscapes when ST:STUDY_SUMMARY compared with conventional 2D monolayers. Using a new genetic screening platform ST:STUDY_SUMMARY we identify MAPK14 (which encodes the protein kinase p38alpha) as a specific ST:STUDY_SUMMARY requirement for HGEC growing in spheroid culture. We show that MAPK14/p38alpha ST:STUDY_SUMMARY has broad roles in programing the phosphoproteome, transcriptome and metabolome ST:STUDY_SUMMARY of HGEC spheroids, yet has negligible impact on monolayer cultures. Using ST:STUDY_SUMMARY single-cell analysis, we show that MAPK14 is specifically required to sustain a ST:STUDY_SUMMARY sub-population of spheroid cells that is enriched in cancer stemness markers. ST:STUDY_SUMMARY MAPK14 also promotes cancer HGEC tumorigenicity in vivo. We conclude that ST:STUDY_SUMMARY spheroid growth of HGEC activates unique biological programs, including p38alpha ST:STUDY_SUMMARY signaling, that are highly relevant to malignant disease pathology and cannot be ST:STUDY_SUMMARY captured using 2D culture models. Our work reveals MAPK14/p38alpha as a new ST:STUDY_SUMMARY dependency and potential therapeutic target in HGEC. The experimental models and ST:STUDY_SUMMARY comprehensive data sets presented here are a resource that will help provide a ST:STUDY_SUMMARY better mechanistic understanding of HGEC biology and facilitate translational ST:STUDY_SUMMARY advances. ST:INSTITUTE University of North Carolina at Chapel Hill ST:LAST_NAME Rushing ST:FIRST_NAME Blake ST:ADDRESS 500 Laureate Way ST:EMAIL blake_rushing@unc.edu ST:PHONE 7042505000 ST:SUBMIT_DATE 2024-06-17 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - S_1 3D vs 2D:2D | genotype:Control | Sample source:Cultured cells RAW_FILE_NAME=S_1 SUBJECT_SAMPLE_FACTORS - S_5 3D vs 2D:2D | genotype:Control | Sample source:Cultured cells RAW_FILE_NAME=S_5 SUBJECT_SAMPLE_FACTORS - S_9 3D vs 2D:2D | genotype:Control | Sample source:Cultured cells RAW_FILE_NAME=S_9 SUBJECT_SAMPLE_FACTORS - S_10 3D vs 2D:2D | genotype:MAPK14 KO | Sample source:Cultured cells RAW_FILE_NAME=S_10 SUBJECT_SAMPLE_FACTORS - S_2 3D vs 2D:2D | genotype:MAPK14 KO | Sample source:Cultured cells RAW_FILE_NAME=S_2 SUBJECT_SAMPLE_FACTORS - S_6 3D vs 2D:2D | genotype:MAPK14 KO | Sample source:Cultured cells RAW_FILE_NAME=S_6 SUBJECT_SAMPLE_FACTORS - S_11 3D vs 2D:3D | genotype:Control | Sample source:Cultured cells RAW_FILE_NAME=S_11 SUBJECT_SAMPLE_FACTORS - S_3 3D vs 2D:3D | genotype:Control | Sample source:Cultured cells RAW_FILE_NAME=S_3 SUBJECT_SAMPLE_FACTORS - S_7 3D vs 2D:3D | genotype:Control | Sample source:Cultured cells RAW_FILE_NAME=S_7 SUBJECT_SAMPLE_FACTORS - S_12 3D vs 2D:3D | genotype:MAPK14 KO | Sample source:Cultured cells RAW_FILE_NAME=S_12 SUBJECT_SAMPLE_FACTORS - S_4 3D vs 2D:3D | genotype:MAPK14 KO | Sample source:Cultured cells RAW_FILE_NAME=S_4 SUBJECT_SAMPLE_FACTORS - S_8 3D vs 2D:3D | genotype:MAPK14 KO | Sample source:Cultured cells RAW_FILE_NAME=S_8 SUBJECT_SAMPLE_FACTORS - B_1 3D vs 2D:- | genotype:- | Sample source:blank RAW_FILE_NAME=B_1 SUBJECT_SAMPLE_FACTORS - B_2 3D vs 2D:- | genotype:- | Sample source:blank RAW_FILE_NAME=B_2 SUBJECT_SAMPLE_FACTORS - B_3 3D vs 2D:- | genotype:- | Sample source:blank RAW_FILE_NAME=B_3 SUBJECT_SAMPLE_FACTORS - B_4 3D vs 2D:- | genotype:- | Sample source:blank RAW_FILE_NAME=B_4 SUBJECT_SAMPLE_FACTORS - SP_1 3D vs 2D:- | genotype:- | Sample source:pooled sample RAW_FILE_NAME=SP_1 SUBJECT_SAMPLE_FACTORS - SP_2 3D vs 2D:- | genotype:- | Sample source:pooled sample RAW_FILE_NAME=SP_2 SUBJECT_SAMPLE_FACTORS - SP_3 3D vs 2D:- | genotype:- | Sample source:pooled sample RAW_FILE_NAME=SP_3 SUBJECT_SAMPLE_FACTORS - SP_4 3D vs 2D:- | genotype:- | Sample source:pooled sample RAW_FILE_NAME=SP_4 #COLLECTION CO:COLLECTION_SUMMARY Endometrial cancer cells were cultured in RPMI-1640 medium (Gibco, 11875093) CO:COLLECTION_SUMMARY supplemented with 10% FBS (Gibco, 26140079) and 1% penicillin/streptomycin CO:COLLECTION_SUMMARY (Gibco, 15140122). Cells were cultured at 37°C with 5% CO2. Cell lines were CO:COLLECTION_SUMMARY tested for mycoplasma contamination with the Venor®GeM qEP kit and were CO:COLLECTION_SUMMARY confirmed negative. For 3D spheroid culture, cells were cultured in ultra-low CO:COLLECTION_SUMMARY attachment (ULA) 96-well plates (Corning, 650970) in DMEM/F-12 medium (Gibco, CO:COLLECTION_SUMMARY 11330032) supplemented with 20 ng/ml human EGF recombinant protein (Gibco, CO:COLLECTION_SUMMARY PHG0314), 20 ng/ml human basic FGF recombinant protein (bFGF) (Gibco, 13256029), CO:COLLECTION_SUMMARY 2% B27 supplement (Gibco, 17504044) and 1% penicillin/streptomycin. For CO:COLLECTION_SUMMARY multicellular 3D spheroid culture, HEC-50B cells were seeded in ULA 96-well CO:COLLECTION_SUMMARY plates, at a seeding density of 2000 cells/well for a duration of 4-12 days. CO:COLLECTION_SUMMARY Media was refreshed every two days by aspirating 50% volume and replenishing CO:COLLECTION_SUMMARY with fresh media. For passaging 3D spheroids, spheroids were pelleted by CO:COLLECTION_SUMMARY centrifugation at 400g for 5 min. Spheroid pellets were then washed with 1x DPBS CO:COLLECTION_SUMMARY (Corning, 21031CV). After aspirating DPBS, spheroids were resuspended in CO:COLLECTION_SUMMARY Accutase (Sigma, AT104) at a density of ˜300 spheroids/ml at 37°C for 5 min CO:COLLECTION_SUMMARY with intermittent agitation for uniform dissociation into single cells. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY MAPK14+/+ and MAPK14-/- HEC-50B cells were cultured as monolayers and as 3D TR:TREATMENT_SUMMARY spheroids for 12 days. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Cells were washed three times with ice cold PBS, then quenched with 1 volume of SP:SAMPLEPREP_SUMMARY -20°C cold Acetonitrile (Fisher, A955-1) and collected with additional 0.75 SP:SAMPLEPREP_SUMMARY volume of ultra-pure H2O (Pierce, PI51140). All sample groups included SP:SAMPLEPREP_SUMMARY biological triplicates. Spheroid extracts were dried by speed vac overnight and SP:SAMPLEPREP_SUMMARY then reconstituted in a volume of 95:5 water:methanol that was proportional to SP:SAMPLEPREP_SUMMARY each sample’s protein concentration. Samples were vortexed for 10 min at 5000 SP:SAMPLEPREP_SUMMARY rpm and then centrifuged for 10 min at 16000 x g at 4°C. Supernatants were SP:SAMPLEPREP_SUMMARY transferred to autosampler vials and an aliquot of 10 mL was taken from each SP:SAMPLEPREP_SUMMARY sample and combined into a single mixture to make a quality control study pool SP:SAMPLEPREP_SUMMARY (QCSP). LC-MS grade water was processed in an identical manner as the study SP:SAMPLEPREP_SUMMARY samples to prepare method blanks. #CHROMATOGRAPHY CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) CH:COLUMN_TEMPERATURE 50 ℃ CH:FLOW_GRADIENT Time(min) Flow Rate %A %B Curve 1. 0 0.4 99.0 1.0 5 2. 1.00 0.4 99.0 1.0 5 3. CH:FLOW_GRADIENT 16.00 0.4 1.0 99.0 5 4. 19.00 0.4 1.0 99.0 5 5. 19.50 0.4 99.0 1.0 5 6. 22.00 CH:FLOW_GRADIENT 0.4 99.0 1.0 5 CH:FLOW_RATE 0.4 mL/min CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% methanol; 0.1% formic acid CH:CHROMATOGRAPHY_TYPE Reversed phase #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF-X Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:MS_COMMENTS Metabolomics data were acquired on a Vanquish UHPLC system coupled to a Q MS:MS_COMMENTS Exactive™ HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher MS:MS_COMMENTS Scientific, San Jose, CA). Metabolites were separated via an HSS T3 C18 column MS:MS_COMMENTS (2.1 × 100 mm, 1.7 µm, Waters Corporation) at 50 °C with binary mobile MS:MS_COMMENTS phase of water (A) and methanol (B), each containing 0.1% formic acid (v/v). The MS:MS_COMMENTS UHPLC linear gradient started from 2% B, and increased to 100% B in 16 min, then MS:MS_COMMENTS held for 4 min, with the flow rate at 400 µL/min. The untargeted data was MS:MS_COMMENTS acquired from 70 to 1050 m/z using the data-dependent acquisition mode. Method MS:MS_COMMENTS blanks and SP injections were placed after every 6 samples (n=3 each). MS:MS_COMMENTS Progenesis QI (version 2.1, Waters Corporation) was used for peak picking, MS:MS_COMMENTS alignment, and normalization. Background signals were filtered out by removing MS:MS_COMMENTS peaks with a fold change less than 3 in the total SP vs the blank injections. MS:MS_COMMENTS Samples were then normalized in Progenesis QI using the “normalize to all” MS:MS_COMMENTS feature. Filtered, normalized data was exported and multivariate analysis was MS:MS_COMMENTS performed using SIMCA 16. MS:ION_MODE POSITIVE MS:MS_RESULTS_FILE ST003263_AN005347_Results.txt UNITS:Normalized data. All data is unitless. Note that masses ending in "m/z" are mass-to-charge values while those ending in "n" are neutral masses Has m/z:Yes Has RT:Yes RT units:Minutes #END