#METABOLOMICS WORKBENCH PKSahu_91_20250924_112933 DATATRACK_ID:6481 STUDY_ID:ST004224 ANALYSIS_ID:AN007029 PROJECT_ID:PR002665 VERSION 1 CREATED_ON September 26, 2025, 6:21 am #PROJECT PR:PROJECT_TITLE DNA Damage induced PKD activation affects GOLPH3 mediated chemoresistance PR:PROJECT_TYPE Targeted Lipidomics PR:PROJECT_SUMMARY The GOLPH3 gene, located on chromosome 5p13 and amplified genomically in several PR:PROJECT_SUMMARY major tumors, encodes a Golgi-associated protein that functions as an PR:PROJECT_SUMMARY independent oncogenic driver. Its mechanism of action remains unclear, and no PR:PROJECT_SUMMARY targeted therapies currently exist for GOLPH3-driven tumors. We find that this PR:PROJECT_SUMMARY oncogene operates as part of an unexpectedly complex multimodular system that PR:PROJECT_SUMMARY incorporates diverse molecular pathways converging into a unified framework that PR:PROJECT_SUMMARY underpins chemoresistance. Under genotoxic stress conditions, GOLPH3 acts on GSL PR:PROJECT_SUMMARY metabolism accelerating conversion of proapoptotic ceramide to pro-survival GSL. PR:PROJECT_SUMMARY This, in concert with a Golgi based network of lipid transfer proteins, promotes PR:PROJECT_SUMMARY the formation of cholesterol-GSL-enriched bioactive lipid rafts at the TGN. PR:PROJECT_SUMMARY These rafts incorporate and activate the SRC-JNK complex that triggers a PR:PROJECT_SUMMARY morphological and functional remodeling of the Golgi complex resulting in the PR:PROJECT_SUMMARY accelerated secretion of survival factors, a common adaptive reaction of tumors PR:PROJECT_SUMMARY subjected to stress. Despite its complexity and functional potency, the whole PR:PROJECT_SUMMARY process is blocked by inhibition of the GSL metabolism. This generates a PR:PROJECT_SUMMARY potential vulnerability in GOLPH3-dependent tumors. Computational approaches PR:PROJECT_SUMMARY show that the above mechanism of action is transcriptionally encoded in human PR:PROJECT_SUMMARY tumors, indicating its clinical relevance and suggesting the use of GSL PR:PROJECT_SUMMARY metabolic blockers as a therapeutic approach to GOLPH3-dependent human tumors. PR:INSTITUTE IEOMI-CNR PR:LAST_NAME Russo PR:FIRST_NAME Domenico PR:ADDRESS Via Pietro Castellino, NAPLES, Napoli, 80131, Italy PR:EMAIL domenico.russo@cnr.it PR:PHONE +393208799450 #STUDY ST:STUDY_TITLE DNA Damage induced PKD activation affects GOLPH3 mediated chemoresistance ST:STUDY_TYPE Lipidomics ST:STUDY_SUMMARY Targeted sphingolipidomic profiling of HeLa cells treated with either Etoposide ST:STUDY_SUMMARY (40 μM) or Doxorubicin (500 nM) for 16 hours revealed increased de novo ST:STUDY_SUMMARY ceramide synthesis, accompanied by elevated levels of downstream complex ST:STUDY_SUMMARY glycosphingolipids (GlcCer, LacCer, and Gb3Cer). Mechanistically, both drugs ST:STUDY_SUMMARY activated PKD2 at the Golgi, which in turn enhanced GOLPH3 activity and promoted ST:STUDY_SUMMARY the diversion of ceramide into complex GSL synthesis. These findings uncover a ST:STUDY_SUMMARY novel regulatory pathway by which cancer cells drive chemoresistance, thereby ST:STUDY_SUMMARY shunting pro-apoptotic ceramide into the synthesis of pro-survival GSLs. ST:INSTITUTE IEOMI-CNR ST:LAST_NAME Russo ST:FIRST_NAME Domenico ST:ADDRESS Via Pietro Castellino, 111, 80131 Napoli NA, Italy ST:EMAIL domenico.russo@cnr.it ST:PHONE +393208799450 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:CELL_STRAIN_DETAILS HeLa-M #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 - Ctrl-1 Sample source:wild-type | Treatment:control RAW_FILE_NAME(RAW file name)=CTRL-1.mzML SUBJECT_SAMPLE_FACTORS - Ctrl-2 Sample source:wild-type | Treatment:control RAW_FILE_NAME(RAW file name)=CTRL-2.mzML SUBJECT_SAMPLE_FACTORS - ETO-1 Sample source:wild-type | Treatment:etoposide RAW_FILE_NAME(RAW file name)=ETO-1.mzML SUBJECT_SAMPLE_FACTORS - ETO-2 Sample source:wild-type | Treatment:etoposide RAW_FILE_NAME(RAW file name)=ETO-2.mzML SUBJECT_SAMPLE_FACTORS - DOXO-1 Sample source:wild-type | Treatment:doxorubicin RAW_FILE_NAME(RAW file name)=Doxo-1.mzML SUBJECT_SAMPLE_FACTORS - DOXO-2 Sample source:wild-type | Treatment:doxorubicin RAW_FILE_NAME(RAW file name)=Doxo-2.mzML #COLLECTION CO:COLLECTION_SUMMARY Cells were washed in ice-cold PBS and collected by scraping. Cells were washed CO:COLLECTION_SUMMARY twice in cold PBS followed by brief centrifugation steps. Samples were processed CO:COLLECTION_SUMMARY immediately for lipid extraction or stored in -80 degree until processed. CO:SAMPLE_TYPE HeLa cells CO:COLLECTION_METHOD Gentle scraping CO:STORAGE_CONDITIONS 4℃ CO:COLLECTION_VIALS Glass vials CO:COLLECTION_TUBE_TEMP 4 #TREATMENT TR:TREATMENT_SUMMARY Etoposide or doxorubicin or DMSO treated HeLa cells were fortified with an TR:TREATMENT_SUMMARY internal standard mix. Lipids were extracted by resuspending the cells in 1 mlof TR:TREATMENT_SUMMARY Isopropanol–Water–Ethylacetate (30:10:60, by vol), sonicated for 30 s, TR:TREATMENT_SUMMARY vortexed and centrifuge for 5 min at 3,000 × g. Organic phase was transferred TR:TREATMENT_SUMMARY to a new glass vial and extraction was repeated once more to enhance the yield. TR:TREATMENT_SUMMARY Lipids were dried under vacuum before LC-MS injections. TR:TREATMENT_COMPOUND Etoposide or Doxorubicin and Internal standards #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Samples were spiked with an internal standard mix and lipids were extracted SP:SAMPLEPREP_SUMMARY twice with an extraction mix consisiting of Isopropanol:Water:Ethylacetate SP:SAMPLEPREP_SUMMARY (30:10:60v/v). Extracted lipids were vacuum concentrated and resuspended in SP:SAMPLEPREP_SUMMARY Mobile Phase B. SP:PROCESSING_STORAGE_CONDITIONS 4℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Samples were analyzed with ABSCIEX QTRAP 4500 in a Targeted MRM mode coupled CH:CHROMATOGRAPHY_SUMMARY with Schimadzu Nexera UPLC using a solvent gradient. Ceramides identity was CH:CHROMATOGRAPHY_SUMMARY acheived through MRM analysis with soft fragmentation. Quantitative analysis is CH:CHROMATOGRAPHY_SUMMARY based on calibration curves generated for ceramide specices. J.Bielawski et.al/ CH:CHROMATOGRAPHY_SUMMARY Methods. 2006 Jun;39(2):82-91. doi: 10.1016/j.ymeth.2006.05.004. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME ABSCIEX QTRAP 4500 CH:COLUMN_NAME Acquity UPLC BEH C8 (100 x 3 mm, 1.7 μm, 130A) CH:SOLVENT_A 100% water; 0.01% formic acid; 5 mM Ammonium Formate CH:SOLVENT_B 57% Methanol, 43% Isopropanol; 0.1% formic acid; 5 mM ammonium formate CH:FLOW_GRADIENT 0.01min: 84%B; 3min: 84.6%B; 7.7min: 85.5%B; 8.7min: 85.7%B; 9.2min: 85.8%B; CH:FLOW_GRADIENT 12-12.20min: 85.9%B; 17min: 88.3%B; 18min: 89%B; 19-21 min: 84%B CH:FLOW_RATE 0.3mL/min CH:COLUMN_TEMPERATURE 30℃ #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME IEOMI AN:DETECTOR_TYPE QTRAP AN:SOFTWARE_VERSION Analyst 1.6.2 #MS MS:INSTRUMENT_NAME ABI Sciex 4500 QTrap MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Data Acquisition is performed by using Analyst 1.6.2 software. Data analysis is MS:MS_COMMENTS performed by Skyline software MS:CAPILLARY_VOLTAGE 5500V MS:DRY_GAS_FLOW 40 MS:FRAGMENT_VOLTAGE 15 MS:FRAGMENTATION_METHOD CID MS:ION_SOURCE_TEMPERATURE 300℃ #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS pmoles MS_METABOLITE_DATA_START Samples Ctrl-1 Ctrl-2 ETO-1 ETO-2 DOXO-1 DOXO-2 Factors Sample source:wild-type | Treatment:control Sample source:wild-type | Treatment:control Sample source:wild-type | Treatment:etoposide Sample source:wild-type | Treatment:etoposide Sample source:wild-type | Treatment:doxorubicin Sample source:wild-type | Treatment:doxorubicin Cer d18:1/16:0 107.7259152 104.8723617 138.382847 123.490385 203.1483884 199.1421146 Cer d18:1/24:0 93.19453159 89.63799135 66.5980354 69.28099217 449.9198739 410.5796792 HexCer d18:1/24:0 94.99930719 110.3380378 110.520713 78.44612624 131.1009017 165.6438801 LacCer d18:1/16:0 81.25775033 71.45093997 99.76912281 101.1863688 122.488462 108.172201 Gb3Cer d18:1/24:0 298.990915 299.9519595 368.7936281 357.0585816 547.3157611 522.88132 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Retentiontime Cer d18:1/16:0 8.3 Cer d18:1/24:0 18.4 HexCer d18:1/24:0 16.5 LacCer d18:1/16:0 6.5 Gb3Cer d18:1/24:0 14.9 METABOLITES_END #END