Highlights d FADS2 promiscuity yields unreported families of fatty acids (i.e., n-8, n-10, and n-12) d n-5 and n-13 fatty acids indicate apocryphal activities of SCD-1 and FADS1 d Unusual fatty acids display selective incorporation into phospholipid subclasses d Distinctive enzyme-substrate interactions revealed in tumor tissue regions

De novo lipogenesis is a well-described androgen receptor (AR)-regulated metabolic pathway that supports prostate cancer tumor growth by providing fuel, membrane material, and steroid hormone precursor. In contrast, our current understanding of lipid supply from uptake of exogenous lipids and its regulation by AR is limited, and exogenous lipids may play a much more significant role in prostate cancer and disease progression than previously thought. By applying advanced automated quantitative fluorescence microscopy, we provide the most comprehensive functional analysis of lipid uptake in cancer cells to date and demonstrate that treatment of ARpositive prostate cancer cell lines with androgens results in significantly increased cellular uptake of fatty acids, cholesterol, and low-density lipoprotein particles. Consistent with a direct, regulatory role of AR in this process, androgenenhanced lipid uptake can be blocked by the AR-antagonist enzalutamide, but is independent of proliferation and cellcycle progression. This work for the first time comprehensively delineates the lipid transporter landscape in prostate cancer cell lines and patient samples by analysis of transcriptomics and proteomics data, including the plasma membrane proteome. We show that androgen exposure or deprivation regulates the expression of multiple lipid transporters in prostate cancer cell lines and tumor xenografts and that mRNA and protein expression of lipid transporters is enhanced in bone metastatic disease when compared with primary, localized prostate cancer. Our findings provide a strong rationale to investigate lipid uptake as a therapeutic cotarget in the fight against advanced prostate cancer in combination with inhibitors of lipogenesis to delay disease progression and metastasis. Implications: Prostate cancer exhibits metabolic plasticity in acquiring lipids from uptake and lipogenesis at different disease stages, indicating potential therapeutic benefit by cotargeting lipid supply.

Background Metabolic reprograming, non-mutational epigenetic changes, increased cell plasticity, and multidrug tolerance are early hallmarks of therapy resistance in cancer. In this temporary, therapy-tolerant state, cancer cells are highly sensitive to ferroptosis, a form of regulated cell death that is caused by oxidative stress through excess levels of iron-dependent peroxidation of polyunsaturated fatty acids (PUFA). However, mechanisms underpinning therapy-induced ferroptosis hypersensitivity remain to be elucidated. Methods We used quantitative single-cell imaging of fluorescent metabolic probes, transcriptomics, proteomics, and lipidomics to perform a longitudinal analysis of the adaptive response to androgen receptor-targeted therapies (androgen deprivation and enzalutamide) in prostate cancer (PCa). Results We discovered that cessation of cell proliferation and a robust reduction in bioenergetic processes were associated with multidrug tolerance and a strong accumulation of lipids. The gain in lipid biomass was fueled by enhanced lipid uptake through cargo non-selective (macropinocytosis, tunneling nanotubes) and cargo-selective mechanisms (lipid transporters), whereas de novo lipid synthesis was strongly reduced. Enzalutamide induced extensive lipid remodeling of all major phospholipid classes at the expense of storage lipids, leading to increased desaturation and acyl chain length of membrane lipids. The rise in membrane PUFA levels enhanced membrane fluidity and lipid peroxidation, causing hypersensitivity to glutathione peroxidase (GPX4) inhibition and ferroptosis. Combination treatments against AR and fatty acid desaturation, lipase activities, or growth medium supplementation with antioxidants or PUFAs altered GPX4 dependence. Conclusions Our work provides mechanistic insight into processes of lipid metabolism that underpin the acquisition of therapy-induced GPX4 dependence and ferroptosis hypersensitivity to standard of care therapies in PCa. It demonstrates novel strategies to suppress the therapy-tolerant state that may have potential to delay and combat resistance to androgen receptor-targeted therapies, a currently unmet clinical challenge of advanced PCa. Since enhanced GPX4 dependence is an adaptive phenotype shared by several types of cancer in response to different therapies, our work might have universal implications for our understanding of metabolic events that underpin resistance to cancer therapies.

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 [...

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The naturally occurring pentacyclic diterpenoid gibberellic acid (1) was used in the generation of a drug-like amide library using parallel-solution-phase synthesis. Prior to the synthesis, a virtual library was generated and prioritized based on drug-like physicochemical parameters such as log P, hydrogen bond donor/acceptor counts, and molecular weight. The structures of the synthesized analogues (2-13) were elucidated following analysis of the NMR, MS, UV, and IR data. Compound 12 afforded crystalline material, and its structure was confirmed by X-ray crystallographic analysis. All compounds were evaluated in vitro for cytotoxicity and deregulation of lipid metabolism in LNCaP prostate cancer cells. While no cytotoxic activity was identified at the concentrations tested, synthesized analogues 3, 5, 7, 10, and 11 substantially reduced cellular uptake of free cholesterol in prostate cancer cells, suggesting a novel role of gibberellic acid derivatives in deregulating cholesterol metabolism.

<p>Figure S1: LNCaP, VCaP and DuCaP cells were grown in charcoal-dextran stripped serum (CSS) for 48 h and treated with increasing concentrations of R1881 or DHT in the presence or absence of Enzalutamide (10 µM) or vehicle (Ctrl) for 48 h. Figure S2: Androgens increased lipid uptake of long-chain fatty acids. LAPC4 cells were grown in CSS for 48 h and treated with 1 nM R1881 or vehicle for 48 h. Figure S3: (A) LNCaP cells were synchronized in G0/G1 by androgen deprivation (CSS for 48 h) followed by treatment with Tunicamycin (1 mg/mL), Hydroxyurea (1 M), or Nocodazole (25 ug/mL) for another 24 h, placing cell cycle blocks in G0/G1, S phase and mitosis, respectively. Figure S4: Oncomine analysis of candidate lipid transporters in (A) Grasso [2], (B) Varambally [3] and (C) LaTulippe datasets [4] comparing gene expression of normal prostate gland versus localized, primary prostate cancer tumor samples. Figure S5: (A) DuCaP (top panel) and VCaP cells (bottom panel) were grown in CSS for 48 h and treated with 10 nM DHT in the absence or presence of Enz (10 µM) or vehicle (Ctrl) for 48 h. Table S1. Primer sequences</p>

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