29Background 30 Metabolic reprograming, non-mutational epigenetic changes, increased cell plasticity and 31 multidrug tolerance are early hallmarks of therapy resistance in cancer. In this temporary, 32 therapy-tolerant state, cancer cells are highly sensitive to ferroptosis, a form of regulated cell 33 death that is caused by oxidative stress through excess levels of iron-dependent peroxidation 34 of polyunsaturated fatty acids (PUFA). However, mechanisms underpinning therapy-induced 35 ferroptosis hypersensitivity remain to be elucidated. 37Methods 38 We used quantitative single cell imaging of fluorescent metabolic probes, transcriptomics, 39 proteomics and lipidomics to perform a longitudinal analysis of the adaptive response to 40 androgen receptor-targeted therapies (androgen deprivation and enzalutamide) in prostate 41 cancer (PCa). 43Results 44 We discovered that cessation of cell proliferation and a robust reduction in bioenergetic 45 processes were associated with multidrug tolerance and a strong accumulation of lipids. The 46 gain in lipid biomass was fueled by enhanced lipid uptake through cargo non-selective 47 (macropinocytosis, tunneling nanotubes) and cargo-selective mechanisms (lipid transporters), 48 whereas de novo lipid synthesis was strongly reduced. Enzalutamide induced extensive lipid 49 remodeling of all major phospholipid classes at the expense of storage lipids, leading to 50 increased desaturation and acyl chain length of membrane lipids. The rise in membrane PUFA 51 levels enhanced membrane fluidity and lipid peroxidation, causing hypersensitivity to 52 glutathione peroxidase (GPX4) inhibition and ferroptosis. Combination treatments against AR 53 and fatty acid desaturation, lipase activities or growth medium supplementation with 54 antioxidants or PUFAs altered GPX4 dependence. Despite multidrug tolerance, PCa cells 55 displayed an enhanced sensitivity to inhibition of lysosomal processing of exogenous lipids, 56 highlighting an increased dependence on lipid uptake in the therapy-tolerant state. 57 58 Conclusions 59 Our work provides mechanistic insight into processes of lipid metabolism that underpin the 60 acquisition of therapy-induced GPX4 dependence and ferroptosis hypersensitivity to standard 61 of care therapies in PCa. It demonstrated novel strategies to suppress the therapy-tolerant state 62 that may have potential to delay and combat resistance to androgen receptor-targeted therapies, 63 a currently unmet clinical challenge of advanced PCa. Since enhanced GPX4 dependence is an 64 adaptive phenotype shared by several types of cancer in response to different therapies, our 65 work might have universal implications for our understanding of metabolic events that 66 underpin resistance to cancer therapies. 67 68 69 Background 74 Despite significant advancements in detection and treatment over the past decades, prostate 75 cancer (PCa) remains the second most commonly diagnosed cancer among men and the third 76 leading cause of cancer mortality in men worldwide [...
Current technologies for fatty acid analysis are overly reliant on the availability of reference libraries, databases and standards. This reliance is hampering the discovery of novel fatty acids and obscuring a full view of lipid metabolism across the kingdoms of life. Recent high-profile discoveries of novel lipids with site(s) of unsaturation inconsistent with canonical lipid metabolism stress the need for new technologies that can provide robust identifications of fatty acids without reliance on prior assumptions. Herein we present advances in liquid chromatography-mass spectrometry for the de novo identification and quantification of fatty acids in lipid extracts. The workflow combines fixed-charge derivatization with ozone-induced dissociation (OzID) for sensitive detection of low-abundant fatty acids with unambiguous assignment of site(s) of unsaturation. Chromatographic alignment of precursor ions with OzID transitions is undertaken using a bespoke Skyline pipeline providing unbiased identification of fatty acids over a relative concentration range spanning more than 4-orders of magnitude. Application of this analytical workflow to lipid extracts from diverse sources including human plasma, cell lines and vernix caseosa has led to the discovery of new fatty acids that point to hitherto undescribed metabolism within the source cell or organism. The description of these “unknown unknowns” particularly in well-studied systems “such as human plasma” points to a substantially great diversity in the lipidome than previously appreciated.
Many families of lipid isomers remain unresolved by contemporary liquid chromatography-mass spectrometry ap-proaches, leading to a significant underestimation of structural diversity within the lipidome. While ion-mobility coupled to mass spectrometry has provided an additional dimension of lipid isomer resolution, some isomers require resolving power beyond the capabilities of conventional platforms. Here we present the application of high-resolution travelling-wave ion mobility for the separation of lipid isomers that differ in (i) the location of a single car-bon-carbon double bond, (ii) the stereochemistry of the double bond (cis or trans) or, for glycerolipids, (iii) the rela-tive substitution of acyl chains on the glycerol backbone (sn-position). Collisional activation following mobility sepa-ration allowed identification of carbon-carbon double bond position and sn-position, enabling confident interpreta-tion of variations in mobility-peak abundance. To demonstrate the applicability of this method, double bond and sn-position isomers of an abundant phosphatidylcholine composition were resolved in extracts from a prostate cancer cell line and identified by comparison to pure isomer reference standards, revealing the presence of six isomers. These findings suggest that ultra-high resolution ion-mobility has broad potential for isomer-resolved lipidomics and is attractive to consider for future integration with other modes of ion-activation, thereby bringing together advanced orthogonal separations and structure elucidation to provide a more complete picture of the lipidome.
Advanced prostate cancer (PCa) is currently incurable, and development of novel treatments requires a greater understanding of key pathways that are altered during PCa progression. We have focussed on fatty acid metabolism, as the major source of energy for PCa cells, and have implicated a monounsaturated fatty acid (MUFA), cis-vaccenic acid (cVA), as a critical regulator of PCa cell homeostasis. Production of cVA requires stearoyl-CoA desaturase 1 (SCD1), the enzyme attributed to general MUFA production, and whose activity is important in a range of cancer types. Nonetheless, the specific role of cVA has not previously been studied, in PCa nor cancer more generally. To elucidate the role of cVA in PCa more precisely, we used SCD1 inhibition to study PCa responses to broad MUFA depletion and specific MUFA supplementation, comparing the responses of cVA with its more common structural isomer oleate. To accomplish these aims, we utilised cell line models of PCa (LNCaP, MR49F and V16D) and a benign prostate cell line (PNT1A) to perform cell viability, lipidomic and flow cytometric assays. In addition, responses in clinically-relevant patient-derived tumor explants were measured by proliferative (Ki67) and cell death (CC3) marker staining. Pharmacological SCD1 inhibition (SCDi; A939572) decreased cell viability and increased cell death in PCa cell lines and patient-derived tumor explants, while only minimally affecting the benign prostate cell line PNT1A. Phenotypically, analysis of mitochondrial function and lipidomic changes under SCDi suggested a role for MUFAs in regulating mitochondrial membrane composition via cardiolipins, and oxidative stress levels. This in turn has the potential to regulate cell death, control tumor burden, and influence overall PCa patient survival. Supplementation of cVA or oleate under SCDi demonstrated that only cVA was successful at rescuing cell viability, and cVA, but not oleate, could rescue changes observed in mitochondrial-related lipids, suggesting that cVA regulation of mitochondrial function was a major determinant of its effect on cell viability. Additionally, cVA alone dose-dependently increased PCa cell viability, implicating cVA as an important oncogenic factor. Together, these data identify cVA as a novel substrate required for PCa cell viability, likely via regulation of mitochondrial homeostasis and associated cell death pathways. In summary, this research has revealed more precise insights into the oncogenic effects of an overlooked MUFA, cVA, in PCa, and may uncover new druggable targets that are more selective, to improve treatment options for advanced PCa. Citation Format: Julia S. Scott, Reuben Young, Swati Irani, Jonas Dehairs, Stephen Blanksby, Johannes V. Swinnen, Zeyad D. Nassar, Lisa M. Butler. A fat lot of good: A novel monounsaturated fatty acid promotes prostate cancer growth and survival [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A031.
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