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Proteomics comparison of ovarian cancer cells with drug administration and tRNA transfection (24 hours)

ABSTRACT: Quantitative proteome analyses of ovarian cancer cells (TOV-112D cells) in the following three conditions were carried out. 1: Treatment with transfection reagent followed by treatment with DMSO 2: Treatment with transfection reagent followed by treatment with 100nM Camptothecin 3: Treatment with transfection reagent and 10nM tRNALeu(TAA) followed by treatment with 100nM Camptothecin

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Takeshi Masuda

PROVIDER: PXD066104 | JPOST Repository | Sat Jul 26 00:00:00 BST 2025

REPOSITORIES: jPOST

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Project description:Ovarian cancer is the leading cause of gynecologic cancer-related deaths, with high-grade serous ovarian cancer (HGSOC) being the most common and lethal subtype. While BRCA1/2 mutations are established biomarkers predicting sensitivity to PARP inhibitors (PARPis), a subset of patients with BRCA-proficient HGSOC also respond to PARPi therapy. However, the molecular mechanisms driving PARPi sensitivity in BRCA-proficient tumors remain poorly understood. We hypothesized that the composition of the PARP1 protein complex and PARylation-mediated signaling contribute to PARPi sensitivity or resistance in BRCA-proficient HGSOC. Viability screening of BRCA-proficient ovarian cancer cell lines identified PARPi-sensitive and PARPi-resistant lines. Chemical proteomics using rucaparib revealed enrichment of PARP1, PARP2, and associated binding partners, with notably higher MSH6 levels in sensitive lines. Co-immunoprecipitation further uncovered distinct PARP1–MSH6–PARP2 complex abundance between sensitive and resistant cells. Targeting of MSH6 via CRISPR and siRNA conferred rucaparib resistance, particularly in sensitive lines. To explore downstream signaling, we performed ADP-ribosylation proteomics using clickable NAD⁺ analogs, revealing distinct PARylation profiles between sensitive and resistant lines. CHAF1A, a known MSH6 interactor and PARP1 substrate, was enriched in PARPi-sensitive cells. MSH6 knockdown led to increased CHAF1A expression in both sensitive and resistant lines, regardless of rucaparib treatment. Importantly, CHAF1A silencing significantly impaired cell viability, especially in A2780 cells, and suppressed mTOR signaling, suggesting that CHAF1A acts downstream of MSH6 to regulate the mTOR axis. Furthermore, co-treatment with mTORC1 inhibitors enhanced the cellular effects of rucaparib in resistant cells, underscoring the therapeutic potential of targeting downstream mTOR effectors to overcome intrinsic resistance. In conclusion, this study identifies a novel PARP1–MSH6–CHAF1A axis that regulates PARPi sensitivity via mTOR signaling in BRCA-proficient ovarian cancer. By integrating chemical proteomics and ADP-ribosylation analysis, we delineate the interplay between PARP1 complex composition and signaling dynamics, highlighting MSH6 and CHAF1A as critical modulators of PARPi response and potential biomarkers to enhance therapeutic efficacy in BRCA-proficient HGSOC. Three tandem mass tag experiments were performed with the same experimental design to analyze three biological replicates. They are described with reporter ion, treatment (DMSO or 10 micromolar Rucaparib), and cell line: 126: DMSO A2780 127N: Rucaparib A2780 127C: DMSO Caov3 128N: Rucaparib Caov3 128C: DMSO OV CAR8 129N: Rucaparib OVCAR8 129C: DMSO SKOV3 130N: Rucaparib SKOV3 130C: DMSO OVCA432 131N: Rucaparib OVCA432 131C: DMSO HEYA8 132N: Rucaparib HEYA8 134N: DMSO Control Pool

Project description:Advanced ovarian cancer is the most lethal gynecologic malignancy in the United States. Currently patients are treated by surgical cytoreductive surgery with the aim of reducing tumor burden to microscopic disease followed by adjuvant combined treatment with a platinum and taxane containing chemotherapy, which affords 80% of patients an initial complete response. However, Abdominal and pelvic recurrence rates are high and response to further chemotherapy is limited. Attempts at introducing biologic therapeutic agents to improve outcome in this disease are ongoing, while prognostic or predictive biomarkers that can stratify patients for treatment are still lacking. Using a 60-mer 22K oligonucleotide-based array comparative genome hybridization (CGH) platform combined with DNA isolated from microdissected tumor tissue samples, Birrer et. al. reported that the amplification of 5q31-35.3 in ovarian cancer cells is a negative prognostic indicator for patients with advanced stage high-grade serous ovarian cancer (HGSC). Further studies showed that fibroblast growth factor 1 (FGF1) located in the amplicon, may be one of the driving genes for ovarian cancer progression (Birrer et. al., 2007). Besides FGF1, located on the same amplicon is one of its receptors fibroblast growth factor receptor 4 (FGFR4), suggesting that it may also be amplified and may be another driving gene involved in ovarian cancer pathogenesis.In this study, we used microarrays to explore and compare gene expression profiles between FGFR4 knock down ovarian cancer cell lines and their corresponding parental cell lines. Two high grade serous ovarian cancer cell lines: SKOV3 and OVCA432 were selected for this study. For each of the cell lines, two different siRNA sequences which both target human FGFR4 were used. While a non-target scramble siRNA sequence was used as control. Ovarian cancer cells were transfected with siRNA using lipofectamine (Invitrogen). RNA extraction was performed 72 hours post-transfection, followed by hybridization on Affymetrix microarrays.

Project description:The actions of environmental toxicants and relevant mixtures in promoting the epigenetic transgenerational inheritance of ovarian disease was investigated with the use of a fungicide, a pesticide mixture, a plastic mixture, dioxin and a hydrocarbon mixture. After transient exposure of an F0 gestating female rat during embryonic gonadal sex determination, the F1 and F3 generation progeny adult onset ovarian disease was assessed. Transgenerational disease phenotypes observed included an increase in cysts resembling human polycystic ovarian disease (PCO) and a decrease in the ovarian primordial follicle pool size resembling primary ovarian insufficiency (POI). The F3 generation granulosa cells were isolated and found to have a transgenerational effect on the transcriptome and epigenome (differential DNA methylation). Epigenetic biomarkers for environmental exposure and associated gene networks were identified. Epigenetic transgenerational inheritance of ovarian disease states was induced by all the different classes of environmental compounds, suggesting a role of environmental epigenetics in ovarian disease etiology. Granulosa cells from large antral follicles were collected and evaluated from F3 generation rats that were ancestrally exposed to one of the five different treatments: Vinclozolin, Pesticide (includes permethrin and DEET), Plastics (includes BPA, DBP and DEHP), Low-dose Plastics (50% of Plastics dose), Dioxin, Hydrocarbon (Jet fuel JP8), or DMSO vehicle as Control. Vinclozolin lineage alterations in differentially DNA methylated regions (DMR) in the granulosa cells was investigated by using a methylated DNA immunoprecipitation (MeDIP) procedure followed by comparative hybridization on a genome wide promoter tiling array (Chip), termed an MeDIP-Chip assay. The DNA fractions from four animals of the same treatment group were pooled to create three different pooled DNA samples from each of the two treatment groups (experimental vs. control). These DNA samples were then used for methylated DNA immunoprecipitation (MeDIP) using Nimblegen microarrays. Each MeDIP sample was then used to preform three different comparative (amplified MeDIP vs. amplified MeDIP) hybridization experiments (3 sub-arrays), each encompassing DNA samples from 24 animls (3 treatment and 3 control groups).

			Action	DRS
	240818_CPTLeu_1.mzML	Mzml
	240818_CPTLeu_1.raw	Raw
	240818_CPTLeu_2.mzML	Mzml
	240818_CPTLeu_2.raw	Raw
	240818_CPTLeu_3.mzML	Mzml

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Proteomics comparison of ovarian cancer cells with drug administration and tRNA transfection (24 hours)

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