ABSTRACT
Resistance to platinum compounds is a major determinant of patient survival in high-grade serous ovarian cancer (HGSOC). To understand mechanisms of platinum resistance and identify potential therapeutic targets in resistant HGSOC, we generated a data resource composed of dynamic (±carboplatin) protein, post-translational modification, and RNA sequencing (RNA-seq) profiles from intra-patient cell line pairs derived from 3 HGSOC patients before and after acquiring platinum resistance. These profiles reveal extensive responses to carboplatin that differ between sensitive and resistant cells. Higher fatty acid oxidation (FAO) pathway expression is associated with platinum resistance, and both pharmacologic inhibition and CRISPR knockout of carnitine palmitoyltransferase 1A (CPT1A), which represents a rate limiting step of FAO, sensitize HGSOC cells to platinum. The results are further validated in patient-derived xenograft models, indicating that CPT1A is a candidate therapeutic target to overcome platinum resistance. All multiomic data can be queried via an intuitive gene-query user interface (https://sites.google.com/view/ptrc-cell-line).
Subject(s)
Carboplatin/therapeutic use , Carnitine O-Palmitoyltransferase/metabolism , Cystadenocarcinoma, Serous/metabolism , Cystadenocarcinoma, Serous/pathology , Genomics , Molecular Targeted Therapy , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Acetyl-CoA Carboxylase/genetics , Acetyl-CoA Carboxylase/metabolism , Animals , Apoptosis/drug effects , Carboplatin/pharmacology , Carnitine O-Palmitoyltransferase/antagonists & inhibitors , Carnitine O-Palmitoyltransferase/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Cystadenocarcinoma, Serous/drug therapy , DNA Damage , Drug Resistance, Neoplasm/drug effects , Fatty Acids/metabolism , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Mice, SCID , Neoplasm Grading , Ovarian Neoplasms/drug therapy , Oxidation-Reduction/drug effects , Oxidative Phosphorylation/drug effects , Phosphoproteins/metabolism , Proteomics , Reactive Oxygen Species/metabolismABSTRACT
Cisplatin (cis-diamminedichloroplatinum(II)) causes crosslinking of DNA at AG and GG sites in cellular DNA, inhibiting replication, and making it a useful anti-cancer drug. Several techniques have been used previously to detect nucleic acid damage but most of these tools are labour-intensive, time-consuming, and/or expensive. Here, we describe a sensitive, robust, and quantitative tool for detecting cisplatin-induced DNA damage by using fluorescent molecular beacon probes (MB). Our results show a decrease of fluorescence in the presence of cisplatin-induced DNA damage, confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The decrease in fluorescence upon damage scales with the number of AG and GG sites, indicating the ability of MB to quantitatively detect DNA damage by cisplatin.
Subject(s)
Antineoplastic Agents/toxicity , Cisplatin/toxicity , DNA Damage , Molecular Probes , Fluorescence , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Ultraviolet A (UVA)-irradiated 4'-hydroxymethyl-4,5',8-trimethyl psoralen (HMT) in the presence of a poly-dT(17) and dA(7) TTA(8) oligonucleotides produces HMT-dT(17) and HMT-dA(7) TTA(8) adducts in aqueous solution. In this article, we determine whether these HMT-dT(17) and HMT-dA(7) TTA(8) adducts can be detected with a molecular beacon (MB) probe. We measure the degree of damage in dT(17) and dA(7) TTA(8) solutions containing UVA-activated HMT via monitoring the decrease in MB fluorescence. Photoproduct formation is confirmed by MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-fight mass spectrometry measurements) and absorption spectroscopy. The MB fluorescence decreases upon UVA irradiation in the presence of HMT with a single-exponential time constants of 114.2 ± 6.5 min for HMT-dT(17) adducts and 677.8 ± 181.8 min for HMT-dA(7) TTA(8) adducts. Our results show that fluorescent MB probes are a selective, robust and accurate tool for detecting UVA-activated HMT-induced DNA damage.
