High-grade serous ovarian cancer (HGSOC) is often sensitive to initial treatment with platinum and taxane combination chemotherapy, but most patients relapse with chemotherapy-resistant disease. To systematically identify genes modulating chemotherapy response, we performed pooled functional genomic screens in HGSOC cell lines treated with cisplatin, paclitaxel, or cisplatin plus paclitaxel. Genes in the intrinsic pathway of apoptosis were among the top candidate resistance genes in both gain-of-function and loss-of-function screens. In an open reading frame overexpression screen, followed by a mini-pool secondary screen, anti-apoptotic genes including BCL2L1 (BCL-XL) and BCL2L2 (BCL-W) were associated with chemotherapy resistance. In a CRISPR-Cas9 knockout screen, loss of BCL2L1 decreased cell survival whereas loss of proapoptotic genes promoted resistance. To dissect the role of individual anti-apoptotic proteins in HGSOC chemotherapy response, we evaluated overexpression or inhibition of BCL-2, BCL-XL, BCL-W, and MCL1 in HGSOC cell lines. Overexpression of anti-apoptotic proteins decreased apoptosis and modestly increased cell viability upon cisplatin or paclitaxel treatment. Conversely, specific inhibitors of BCL-XL, MCL1, or BCL-XL/BCL-2, but not BCL-2 alone, enhanced cell death when combined with cisplatin or paclitaxel. Anti-apoptotic protein inhibitors also sensitized HGSOC cells to the poly (ADP-ribose) polymerase inhibitor olaparib. These unbiased screens highlight anti-apoptotic proteins as mediators of chemotherapy resistance in HGSOC, and support inhibition of BCL-XL and MCL1, alone or combined with chemotherapy or targeted agents, in treatment of primary and recurrent HGSOC. Implications: Anti-apoptotic proteins modulate drug resistance in ovarian cancer, and inhibitors of BCL-XL or MCL1 promote cell death in combination with chemotherapy.
Imaging approaches that track biological molecules within cells are essential tools in modern biochemistry. Lipids are particularly challenging to visualize, as they are not directly genetically encoded. Phospholipids, the most abundant subgroup of lipids, are structurally diverse and accomplish many cellular functions, acting as major structural components of membranes and as signaling molecules that regulate cell growth, division, apoptosis, cytoskeletal dynamics, and numerous other physiological processes. Cells regulate the abundance, and therefore bioactivity, of phospholipids by modulating the activities of their biosynthetic enzymes. Thus, techniques that enable monitoring of flux through individual lipid biosynthetic pathways can provide key functional information. For example, the choline analogue propargylcholine (ProCho) can report on de novo biosynthesis of phosphatidylcholine by conversion to an alkynyl lipid that can be imaged following click chemistry tagging with an azido fluorophore. We report that ProCho is also a substrate of phospholipase D enzymes-which normally hydrolyze phosphatidylcholine to generate the lipid second messenger phosphatidic acid-in a transphosphatidylation reaction, generating the identical alkynyl lipid. By controlling the activities of phosphatidylcholine biosynthesis and phospholipase D enzymes, we establish labeling conditions that enable this single probe to selectively report on two different biosynthetic pathways. Just as nature exploits the economy of common metabolic intermediates to efficiently diversify biosynthesis, so can biochemists in interrogating such pathways with careful probe design. We envision that ProCho's ability to report on multiple metabolic pathways will enable studies of membrane dynamics and improve our understanding of the myriad roles that lipids play in cellular homeostasis.
<div>Abstract<p>High-grade serous ovarian cancer (HGSOC) is often sensitive to initial treatment with platinum and taxane combination chemotherapy, but most patients relapse with chemotherapy-resistant disease. To systematically identify genes modulating chemotherapy response, we performed pooled functional genomic screens in HGSOC cell lines treated with cisplatin, paclitaxel, or cisplatin plus paclitaxel. Genes in the intrinsic pathway of apoptosis were among the top candidate resistance genes in both gain-of-function and loss-of-function screens. In an open reading frame overexpression screen, followed by a mini-pool secondary screen, anti-apoptotic genes including <i>BCL2L1</i> (BCL-XL) and <i>BCL2L2</i> (BCL-W) were associated with chemotherapy resistance. In a CRISPR-Cas9 knockout screen, loss of <i>BCL2L1</i> decreased cell survival whereas loss of proapoptotic genes promoted resistance. To dissect the role of individual anti-apoptotic proteins in HGSOC chemotherapy response, we evaluated overexpression or inhibition of BCL-2, BCL-XL, BCL-W, and MCL1 in HGSOC cell lines. Overexpression of anti-apoptotic proteins decreased apoptosis and modestly increased cell viability upon cisplatin or paclitaxel treatment. Conversely, specific inhibitors of BCL-XL, MCL1, or BCL-XL/BCL-2, but not BCL-2 alone, enhanced cell death when combined with cisplatin or paclitaxel. Anti-apoptotic protein inhibitors also sensitized HGSOC cells to the poly (ADP-ribose) polymerase inhibitor olaparib. These unbiased screens highlight anti-apoptotic proteins as mediators of chemotherapy resistance in HGSOC, and support inhibition of BCL-XL and MCL1, alone or combined with chemotherapy or targeted agents, in treatment of primary and recurrent HGSOC.</p>Implications:<p>Anti-apoptotic proteins modulate drug resistance in ovarian cancer, and inhibitors of BCL-XL or MCL1 promote cell death in combination with chemotherapy.</p></div>
<p>Figure S1A. Cisplatin and paclitaxel sensitivity in ovarian cancer cell lines Figure S1B. Cell line growth curves in ORF screen Figure S1C. Candidate resistance genes scoring in {greater than or equal to}2 conditions in primary ORF screen Figure S2A. Expression of MDR1, encoded by ABCB1 Figure S2B. Overexpression of ABCB1 and paclitaxel response Figure S2C. Overexpression of ABCB1 and paclitaxel response in HGSOC cell lines Figure S2D. Effect of ABCB1 overexpression on cell growth with paclitaxel treatment Figure S2E. Effect of MDR1/P-glycoprotein inhibitor elacridar on paclitaxel response Figure S2F. Overexpression of ABCB1 and colony formation with paclitaxel treatment and effect of elacridar on colony formation Figure S3A. Cell line growth curves in mini-pool overexpression secondary screen Figure S3B. Mini-pool overexpression secondary screen Figure S3C. Top-ranked genes by LFC in mini-pool overexpression secondary screen Figure S4A. Cell line growth curves in CRISPR-Cas9 screen Figure S4B. CRISPR-Cas9 screen data from cisplatin+paclitaxel "low-dose" combination Figure S5A. Expression of BCL-XL and BCL-W Figure S5B. Overexpression of BCL2L1 (BCL-XL) or BCL2L2 (BCL-W) and paclitaxel response in HGSOC cell lines Figure S5C. Effect of overexpressing anti-apoptotic proteins on apoptotic priming Figure S5D. Expression of BCL-2 and MCL1 Figure S5E. Overexpression of BCL2 or MCL1 and apoptosis induction Figure S5F. Overexpression of BCL2 or MCL1 and paclitaxel or cisplatin response Figure S6. Cell cycle analysis of HGSOC cells overexpressing anti-apoptotic proteins Figure S7A. Genomic alterations in anti-apoptotic genes in primary ovarian cancer Figure S7B. Copy-number alterations of anti-apoptotic genes in primary HGSOC Figure S7C. Copy-number alterations of pro-apoptotic genes in primary HGSOC Figure S7D. Copy number and expression of anti-apoptotic genes in primary HGSOC Figure S7E. MCL1 and BCL2L1 are focally amplified in HGSOC Figure S7F. Progression-free interval of patients with primary HGSOC with focal amplifications of BCL2L1 or MCL1 Figure S8A. Copy number alterations of anti-apoptotic genes in ovarian cancer cell lines Figure S8B. Expression of anti-apoptotic genes in ovarian cancer cell lines Figure S9A. Expression of anti-apoptotic genes in platinum-sensitive and -resistant HGSOC Figure S9B. Expression of anti-apoptotic genes in paired platinum-sensitive and- resistant HGSOC Figure S10A. Effects of single-agent anti-apoptotic protein inhibitors on ovarian cancer cell lines Figure S10B. BH3 profiling of HGSOC cell lines with a panel of pro-apoptotic peptides</p>
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.