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Figure S1. AUY922 treatment induces the expression of proteins involved in fatty acid. metabolism and increases PCa cell mitochondrial mass and cellular ROS. Figure S2. Combination treatment of AUY922 with antagonists of fatty acid oxidation (FAO) and oxidative phosphorylation reveals functional interactions. Figure S3. AUY922 and PEX act synergistically on PCa cells. Figure S4. AUY922 and PEX act synergistically on PNT1 cells. Figure S5. PEX decreased fatty acid oxidation in AUY922 treated PCa cells. Figure S6. The effect of AUY922 and PEX combination treatment on AKT, ERK and S6 signaling pathways. Figure S7. AUY922 and PEX combination treatment induces apoptosis in 22RV1 and PNT1 cells. Figure S8. AUY922 and PEX combination treatment induces apoptosis in cultured primary prostate tumors.

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HSP90 is a molecular chaperone required for stabilization and activation of hundreds of client proteins, including many known oncoproteins. AUY922 (luminespib), a new-generation HSP90 inhibitor, exhibits potent preclinical efficacy against several cancer types including prostate cancer. However, clinical use of HSP90 inhibitors for prostate cancer has been limited by toxicity and treatment resistance. Here, we aimed to design an effective combinatorial therapeutic regimen that utilizes subtoxic doses of AUY922, by identifying potential survival pathways induced by AUY922 in clinical prostate tumors. We conducted a proteomic analysis of 30 patient-derived explants (PDE) cultured in the absence and presence of AUY922, using quantitative mass spectrometry. AUY922 significantly increased the abundance of proteins involved in oxidative phosphorylation and fatty acid metabolism in the PDEs. Consistent with these findings, AUY922-treated prostate cancer cell lines exhibited increased mitochondrial mass and activated fatty acid metabolism processes. We hypothesized that activation of fatty acid oxidation is a potential adaptive response to AUY922 treatment and that cotargeting this process will sensitize prostate cancer cells to HSP90 inhibition. Combination treatment of AUY922 with a clinical inhibitor of fatty acid oxidation, perhexiline, synergistically decreased viability of several prostate cancer cell lines, and had significant efficacy in PDEs. The novel drug combination treatment induced cell-cycle arrest and apoptosis, and attenuated the heat shock response, a known mediator of HSP90 treatment resistance. This combination warrants further preclinical and clinical investigation as a novel strategy to overcome resistance to HSP90 inhibition.Implications:Metabolic pathways induced in tumor cells by therapeutic agents may be critical, but targetable, mediators of treatment resistance.

S1: Pretreatment of PC-3 and C4-2B cells with a FA Mix alters the response to palmitate lipotoxicity and serum-starvation. S2: Dose response assessment of palmitate supplementation on cell viability. S3: Pretreatment with the DGAT1 inhibitor AZD3988 does not affect PC-3 cell growth.

Prostate cancer cells exhibit altered cellular metabolism but, notably, not the hallmarks of Warburg metabolism. Prostate cancer cells exhibit increased de novo synthesis of fatty acids (FA); however, little is known about how extracellular FAs, such as those in the circulation, may support prostate cancer progression. Here, we show that increasing FA availability increased intracellular triacylglycerol content in cultured patient-derived tumor explants, LNCaP and C4-2B spheroids, a range of prostate cancer cells (LNCaP, C4-2B, 22Rv1, PC-3), and prostate epithelial cells (PNT1). Extracellular FAs are the major source (∼83%) of carbons to the total lipid pool in all cell lines, compared with glucose (∼13%) and glutamine (∼4%), and FA oxidation rates are greater in prostate cancer cells compared with PNT1 cells, which preferentially partitioned extracellular FAs into triacylglycerols. Because of the higher rates of FA oxidation in C4-2B cells, cells remained viable when challenged by the addition of palmitate to culture media and inhibition of mitochondrial FA oxidation sensitized C4-2B cells to palmitate-induced apoptosis. Whereas in PC-3 cells, palmitate induced apoptosis, which was prevented by pretreatment of PC-3 cells with FAs, and this protective effect required DGAT-1–mediated triacylglycerol synthesis. These outcomes highlight for the first-time heterogeneity of lipid metabolism in prostate cancer cells and the potential influence that obesity-associated dyslipidemia or host circulating has on prostate cancer progression.Implications:Extracellular-derived FAs are primary building blocks for complex lipids and heterogeneity in FA metabolism exists in prostate cancer that can influence tumor cell behavior.

Fatty liver is characterised by the expansion of lipid droplets and is associated with the development of many metabolic diseases, including insulin resistance, dyslipidaemia and cardiovascular disease. We assessed the morphology of hepatic lipid droplets and performed quantitative proteomics in lean, glucose-tolerant mice compared to high-fat diet (HFD) fed mice that displayed hepatic steatosis and glucose intolerance as well as high-starch diet (HStD) fed mice who exhibited similar levels of hepatic steatosis but remained glucose tolerant. Both HFD and HStD-fed mice had more and larger lipid droplets than Chow-fed animals. We observed striking differences in liver lipid droplet proteomes of HFD and HStD-fed mice compared to Chow-fed mice, with fewer differences between HFD and HStD. Taking advantage of our diet strategy, we identified a fatty liver lipid droplet proteome consisting of proteins common in HFD- and HStD-fed mice. Likewise, a proteome associated with glucose tolerance that included proteins common in Chow and HStD but not HFD-fed mice was identified. Notably, glucose intolerance was associated with changes in the ratio of adipose triglyceride lipase (ATGL) to perilipin 5 (PLIN5) in the lipid droplet proteome, suggesting dysregulation of neutral lipid homeostasis in glucose-intolerant fatty liver, which supports bioactive lipid synthesis and impairs hepatic insulin action. We conclude that our novel dietary approach uncouples ectopic lipid burden from insulin resistance-associated changes in the hepatic lipid droplet proteome.

Prostate tumours are highly reliant on lipids for energy, growth and survival. Activity of the androgen receptor (AR) is associated with reprogramming of lipid metabolic processes in prostate cancer, although the molecular underpinnings of this relationship remain to be fully elucidated. Here, we identified Acyl-CoA Synthetase Medium Chain Family Members 1 and 3 (ACSM1 and ACSM3) as AR-regulated mediators of prostate cancer metabolism and growth. ACSM1 and ACSM3 are upregulated in prostate tumours compared to non-malignant tissues and other cancer types. Both enzymes enhanced proliferation and protected PCa cells from deathin vitro, while silencing ACSM3 led to reduced tumour growth in an orthotopic xenograft model. We show that ACSM1 and ACSM3 are major regulators of the PCa lipidome and enhance energy production via fatty acid oxidation. Metabolic dysregulation caused by loss of ACSM1/3 led to mitochondrial oxidative stress, lipid peroxidation and cell death by ferroptosis. Conversely, over-expression of ACSM1/3 enabled PCa cells to survive toxic doses of medium chain fatty acids and promoted resistance to ferroptosis-inducing drugs and AR antagonists. Collectively, these studies uncover a new link between AR and lipid metabolism and position ACSM1 and ACSM3 as key players in prostate cancer progression and therapy resistance.

Cancer cells undergo drastic metabolic adaptions to cover increased bioenergetic needs, contributing to resistance to therapies. This includes a higher demand for cholesterol, which often coincides with elevated cholesterol uptake from low-density lipoproteins (LDL) and overexpression of the LDL receptor in many cancers. This implies the need for cancer cells to accommodate an increased delivery of LDL along the endocytic pathway to late endosomes/lysosomes (LE/Lys), providing a rapid and effective distribution of LDL-derived cholesterol from LE/Lys to other organelles for cholesterol to foster cancer growth and spread. LDL-cholesterol exported from LE/Lys is facilitated by Niemann–Pick Type C1/2 (NPC1/2) proteins, members of the steroidogenic acute regulatory-related lipid transfer domain (StARD) and oxysterol-binding protein (OSBP) families. In addition, lysosomal membrane proteins, small Rab GTPases as well as scaffolding proteins, including annexin A6 (AnxA6), contribute to regulating cholesterol egress from LE/Lys. Here, we summarize current knowledge that links upregulated activity and expression of cholesterol transporters and related proteins in LE/Lys with cancer growth, progression and treatment outcomes. Several mechanisms on how cellular distribution of LDL-derived cholesterol from LE/Lys influences cancer cell behavior are reviewed, some of those providing opportunities for treatment strategies to reduce cancer progression and anticancer drug resistance.

INTRODUCTION: Altered lipid metabolism and subsequent changes in cellular lipid composition have been observed in prostate cancer cells, are associated with poor clinical outcome, and are promising targets for metabolic therapies. This study reports for the first time on the synthesis of a phospholipid radiotracer based on the phospholipid 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine (PC44:12) to allow tracking of polyunsaturated lipid tumor uptake via PET imaging. This tracer may aid in the development of strategies to modulate response to therapies targeting lipid metabolism in prostate cancer. METHODS: Lipidomics analysis of prostate tumor explants and LNCaP tumor cells were used to identify PC44:12 as a potential phospholipid candidate for radiotracer development. Synthesis of phosphocholine precursor and non-radioactive standard were optimised using click chemistry. The biodistribution of a fluorine-18 labeled analogue, N-{[4-(2-[18F]fluoroethyl)-2,3,4-triazol-1-yl]methyl}-1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine ([18F]2) was determined in LNCaP prostate tumor-bearing NOD SCID gamma mice by ex vivo biodistribution and PET imaging studies and compared to biodistribution of [18F]fluoromethylcholine. RESULTS: [18F]2 was produced with a decay-corrected yield of 17.8 ± 3.7% and an average radiochemical purity of 97.00 ± 0.89% (n = 6). Molar activity was 85.1 ± 3.45 GBq/μmol (2300 ± 93 mCi/μmol) and the total synthesis time was 2 h. Ex vivo biodistribution data demonstrated high liver uptake (41.1 ± 9.2%ID/g) and high splenic uptake (10.9 ± 9.1%ID/g) 50 min post-injection. Ex vivo biodistribution showed low absolute tumor uptake of [18F]2 (0.8 ± 0.3%ID/g). However, dynamic PET imaging demonstrated an increase over time of the relative tumor-to-muscle ratio with a peak of 2.8 ± 0.5 reached 1 h post-injection. In contrast, dynamic PET of [18F]fluoromethylcholine demonstrated no increase in tumor-to-muscle ratios due to an increase in both tumor and muscle over time. Absolute uptake of [18F]fluoromethylcholine was higher and peaked at 60 min post injection (2.25 ± 0.29%ID/g) compared to [18F]2 (1.44 ± 0.06%ID/g) during the 1 h dynamic scan period. CONCLUSIONS AND ADVANCES IN KNOWLEDGE: This study demonstrates the ability to radiolabel phospholipids and indicates the potential to monitor the in vivo distribution of phospholipids using fluorine-18 based PET. ispartof: NUCLEAR MEDICINE AND BIOLOGY vol:93 pages:37-45 ispartof: location:United States status: published

HSP90 is a molecular chaperone required for stabilization and activation of hundreds of client proteins, including many known oncoproteins. AUY922 (luminespib), a new-generation HSP90 inhibitor, exhibits potent preclinical efficacy against several cancer types including prostate cancer. However, clinical use of HSP90 inhibitors for prostate cancer has been limited by toxicity and treatment resistance. Here, we aimed to design an effective combinatorial therapeutic regimen that utilizes subtoxic doses of AUY922, by identifying potential survival pathways induced by AUY922 in clinical prostate tumors. We conducted a proteomic analysis of 30 patient-derived explants (PDE) cultured in the absence and presence of AUY922, using quantitative mass spectrometry. AUY922 significantly increased the abundance of proteins involved in oxidative phosphorylation and fatty acid metabolism in the PDEs. Consistent with these findings, AUY922-treated prostate cancer cell lines exhibited increased mitochondrial mass and activated fatty acid metabolism processes. We hypothesized that activation of fatty acid oxidation is a potential adaptive response to AUY922 treatment and that cotargeting this process will sensitize prostate cancer cells to HSP90 inhibition. Combination treatment of AUY922 with a clinical inhibitor of fatty acid oxidation, perhexiline, synergistically decreased viability of several prostate cancer cell lines, and had significant efficacy in PDEs. The novel drug combination treatment induced cell-cycle arrest and apoptosis, and attenuated the heat shock response, a known mediator of HSP90 treatment resistance. This combination warrants further preclinical and clinical investigation as a novel strategy to overcome resistance to HSP90 inhibition. Implications: Metabolic pathways induced in tumor cells by therapeutic agents may be critical, but targetable, mediators of treatment resistance.

Adipose tissue is essential for metabolic homeostasis, balancing lipid storage and mobilization based on nutritional status. This is coordinated by insulin, which triggers kinase signaling cascades to modulate numerous metabolic proteins, leading to increased glucose uptake and anabolic processes like lipogenesis. Given recent evidence that glucose is dispensable for adipocyte respiration, we sought to test whether glucose is necessary for insulin-stimulated anabolism. Examining lipogenesis in cultured adipocytes, glucose was essential for insulin to stimulate the synthesis of fatty acids and glyceride–glycerol. Importantly, glucose was dispensable for lipogenesis in the absence of insulin, suggesting that distinct carbon sources are used with or without insulin. Metabolic tracing studies revealed that glucose was required for insulin to stimulate pathways providing carbon substrate, NADPH, and glycerol 3-phosphate for lipid synthesis and storage. Glucose also displaced leucine as a lipogenic substrate and was necessary to suppress fatty acid oxidation. Together, glucose provided substrates and metabolic control for insulin to promote lipogenesis in adipocytes. This contrasted with the suppression of lipolysis by insulin signaling, which occurred independently of glucose. Given previous observations that signal transduction acts primarily before glucose uptake in adipocytes, these data are consistent with a model whereby insulin initially utilizes protein phosphorylation to stimulate lipid anabolism, which is sustained by subsequent glucose metabolism. Consequently, lipid abundance was sensitive to glucose availability, both during adipogenesis and in Drosophila flies in vivo. Together, these data highlight the importance of glucose metabolism to support insulin action, providing a complementary regulatory mechanism to signal transduction to stimulate adipose anabolism.

Glutamine is a conditionally essential nutrient for many cancer cells, but it remains unclear how consuming glutamine in excess of growth requirements confers greater fitness to glutamine-addicted cancers. By contrasting two breast cancer subtypes with distinct glutamine dependencies, we show that glutamine-indispensable triple-negative breast cancer (TNBC) cells rely on a non-canonical glutamine-to-glutamate overflow, with glutamine carbon routed once through the TCA cycle. Importantly, this single-pass glutaminolysis increases TCA cycle fluxes and replenishes TCA cycle intermediates in TNBC cells, a process that achieves net oxidation of glucose but not glutamine. The coupling of glucose and glutamine catabolism appears hard-wired via a distinct TNBC gene expression profile biased to strip and then sequester glutamine nitrogen, but hampers the ability of TNBC cells to oxidise glucose when glutamine is limiting. Our results provide a new understanding of how metabolically rigid TNBC cells are sensitive to glutamine deprivation and a way to select vulnerable TNBC subtypes that may be responsive to metabolic-targeted therapies.

Background Both changes in circulating lipids represented by a validated poor prognostic 3-lipid signature (3LS) and somatic tumour genetic aberrations are individually associated with worse clinical outcomes in men with metastatic castration-resistant prostate cancer (mCRPC). A key question is how the lipid environment and the cancer genome are interrelated in order to exploit this therapeutically. We assessed the association between the poor prognostic 3-lipid signature (3LS), somatic genetic aberrations and clinical outcomes in mCRPC. Methods We performed plasma lipidomic analysis and cell-free DNA (cfDNA) sequencing on 106 men with mCRPC commencing docetaxel, cabazitaxel, abiraterone or enzalutamide (discovery cohort) and 94 men with mCRPC commencing docetaxel (validation cohort). Differences in lipid levels between men ± somatic genetic aberrations were assessed with t-tests. Associations between the 3LS and genetic aberrations with overall survival (OS) were examined using Kaplan-Meier methods and Cox proportional hazard models. Results The 3LS was associated with shorter OS in the discovery (hazard ratio [HR] 2.15, 95% confidence interval [CI] 1.4-3.3, p < 0.001) and validation cohorts (HR 2.32, 95% CI 1.59–3.38, p < 0.001). Elevated plasma sphingolipids were associated with AR, TP53, RB1 and PI3K aberrations (p < 0.05). Men with both the 3LS and aberrations in AR, TP53, RB1 or PI3K had shorter OS than men with neither in both cohorts (p ≤ 0.001). The presence of 3LS and/or genetic aberration was independently associated with shorter OS for men with AR, TP53, RB1 and PI3K aberrations (p < 0.02). Furthermore, aggressive-variant prostate cancer (AVPC), defined as 2 or more aberrations in TP53, RB1 and/or PTEN, was associated with elevated sphingolipids. The combination of AVPC and 3LS predicted for a median survival of ~12 months. The relatively small sample size of the cohorts limits clinical applicability and warrants future studies. Conclusions Elevated circulating sphingolipids were associated with AR, TP53, RB1, PI3K and AVPC aberrations in mCRPC, and the combination of lipid and genetic abnormalities conferred a worse prognosis. These findings suggest that certain genotypes in mCRPC may benefit from metabolic therapies.

Prostate cancer cells exhibit altered cellular metabolism but, notably, not the hallmarks of Warburg metabolism. Prostate cancer cells exhibit increased de novo synthesis of fatty acids (FA); however, little is known about how extracellular FAs, such as those in the circulation, may support prostate cancer progression. Here, we show that increasing FA availability increased intracellular triacylglycerol content in cultured patient-derived tumor explants, LNCaP and C4-2B spheroids, a range of prostate cancer cells (LNCaP, C4-2B, 22Rv1, PC-3), and prostate epithelial cells (PNT1). Extracellular FAs are the major source (∼83%) of carbons to the total lipid pool in all cell lines, compared with glucose (∼13%) and glutamine (∼4%), and FA oxidation rates are greater in prostate cancer cells compared with PNT1 cells, which preferentially partitioned extracellular FAs into triacylglycerols. Because of the higher rates of FA oxidation in C4-2B cells, cells remained viable when challenged by the addition of palmitate to culture media and inhibition of mitochondrial FA oxidation sensitized C4-2B cells to palmitate-induced apoptosis. Whereas in PC-3 cells, palmitate induced apoptosis, which was prevented by pretreatment of PC-3 cells with FAs, and this protective effect required DGAT-1–mediated triacylglycerol synthesis. These outcomes highlight for the first-time heterogeneity of lipid metabolism in prostate cancer cells and the potential influence that obesity-associated dyslipidemia or host circulating has on prostate cancer progression. Implications: Extracellular-derived FAs are primary building blocks for complex lipids and heterogeneity in FA metabolism exists in prostate cancer that can influence tumor cell behavior.

Alterations to the androgen receptor (AR) signalling axis and cellular metabolism are hallmarks of prostate cancer. This study provides insight into both hallmarks by uncovering a novel link between AR and the pentose phosphate pathway (PPP). Specifically, we identify 6-phosphogluoconate dehydrogenase (6PGD) as an androgen-regulated gene that is upregulated in prostate cancer. AR increased the expression of 6PGD indirectly via activation of sterol regulatory element binding protein 1 (SREBP1). Accordingly, loss of 6PGD, AR or SREBP1 resulted in suppression of PPP activity as revealed by 1,2-13C2 glucose metabolic flux analysis. Knockdown of 6PGD also impaired growth and elicited death of prostate cancer cells, at least in part due to increased oxidative stress. We investigated the therapeutic potential of targeting 6PGD using two specific inhibitors, physcion and S3, and observed substantial anti-cancer activity in multiple models of prostate cancer, including aggressive, therapy-resistant models of castration-resistant disease as well as prospectively collected patient-derived tumour explants. Targeting of 6PGD was associated with two important tumour-suppressive mechanisms: first, increased activity of the AMP-activated protein kinase (AMPK), which repressed anabolic growth-promoting pathways regulated by acetyl-CoA carboxylase 1 (ACC1) and mammalian target of rapamycin complex 1 (mTORC1); and second, enhanced AR ubiquitylation, associated with a reduction in AR protein levels and activity. Supporting the biological relevance of positive feedback between AR and 6PGD, pharmacological co-targeting of both factors was more effective in suppressing the growth of prostate cancer cells than single-agent therapies. Collectively, this work provides new insight into the dysregulated metabolism of prostate cancer and provides impetus for further investigation of co-targeting AR and the PPP as a novel therapeutic strategy.

Fatty acid metabolism is known to support tumorigenesis and disease progression as well as treatment resistance through enhanced lipid synthesis, storage and catabolism. More recently, the role of membrane fatty acid composition, for example, ratios of saturated, monounsaturated and polyunsaturated fatty acids, in promoting cell survival while limiting lipotoxicity and ferroptosis has been increasingly appreciated. Alongside these insights, it has become clear that tumour cells exhibit plasticity with respect to fatty acid metabolism, responding to extratumoural and systemic metabolic signals, such as obesity and cancer therapeutics, to promote the development of aggressive, treatment-resistant disease. Here, we describe cellular fatty acid metabolic changes that are connected to therapy resistance and contextualize obesity-associated changes in host fatty acid metabolism that likely influence the local tumour microenvironment to further modify cancer cell behaviour while simultaneously creating potential new vulnerabilities.

Alterations to androgen receptor (AR) signalling and cellular metabolism are hallmarks of prostate cancer. This study uncovers a novel link between AR and the pentose phosphate pathway (PPP) through 6-phosphogluoconate dehydrogenase (6PGD), an androgen-regulated gene that is upregulated in prostate cancer. Knockdown of 6PGD impairs growth and elicits death of prostate cancer cells, at least in part due to oxidative stress. Targeting 6PGD using 2 specific inhibitors, physcion and S3, was efficacious in multiple models of prostate cancer, including aggressive castration-resistant models. Importantly, S3 also suppressed proliferation of clinical patient-derived explants (PDEs). Mechanistically, 6PGD decreased expression and activity of AR in cell lines and PDEs, revealing a novel positive feedback loop between these factors. The enhanced efficacy of co-targeting AR and 6PGD further supported the biological relevance of this feedback. This work provides insight into the dysregulated metabolism of prostate cancer and supports investigation of co-targeting AR and the PPP.

The prostate is surrounded by periprostatic adipose tissue (PPAT), the thickness of which has been associated with more aggressive prostate cancer (PCa). There are limited data regarding the functional characteristics of PPAT, how it compares to subcutaneous adipose tissue (SAT), and whether in a setting of localized PCa, these traits are altered by obesity or disease aggressiveness. PPAT and SAT were collected from 60 men (age: 42&ndash, 78 years, BMI: 21.3&ndash, 35.6 kg/m2) undergoing total prostatectomy for PCa. Compared to SAT, adipocytes in PPAT were smaller, had the same basal rates of fatty acid release (lipolysis) yet released less polyunsaturated fatty acid species, and were more sensitive to isoproterenol-stimulated lipolysis. Basal lipolysis of PPAT was increased in men diagnosed with less aggressive PCa (Gleason score (GS) &le, 3 + 4) compared to men with more aggressive PCa (GS &ge, 4 + 3) but no other measured adipocyte parameters related to PCa aggressiveness. Likewise, there was no difference in PPAT lipid biology between lean and obese men. In conclusion, lipid biological features of PPAT do differ from SAT, however, we did not observe any meaningful difference in ex vivo PPAT biology that is associated with PCa aggressiveness or obesity. As such, our findings do not support a relationship between altered PCa behavior in obese men and the metabolic reprogramming of PPAT.

Obese men have lower serum prostate-specific antigen (PSA) than comparably aged lean men, but the underlying mechanism remains unclear. The aim of this study was to determine the effect of obesity on PSA and the potential contributing mechanisms. A cohort of 1195 men aged 35 years and over at recruitment, with demographic, anthropometric (BMI, waist circumference (WC)) and serum hormone (serum testosterone, estradiol (E2)) PSA and hematology assessments obtained over two waves was assessed. Men with a history of prostate cancer or missing PSA were excluded, leaving 970 men for the final analysis. Mixed-effects regressions and mediation analyses adjusting for hormonal and volumetric factors explore the potential mechanisms relating obesity to PSA. After adjusting for age, PSA levels were lower in men with greater WC (P = 0.001). In a multivariable model including WC, age, E2/testosterone and PlasV as predictors, no statistically significant associations were observed between with PSA and either WC (P = 0.36) or PlasV (P = 0.49), while strong associations were observed with both E2/testosterone (P P P = 0.31), while when E2/testosterone is a mediator, the ACME explained roughly 50% of the effect (P

Breast cancer (BrCa) metabolism is geared toward biomass synthesis and maintenance of reductive capacity. Changes in glucose and glutamine metabolism in BrCa have been widely reported, yet the contribution of fatty acids (FAs) in BrCa biology remains to be determined. We recently reported that adipocyte coculture alters MCF‐7 and MDA‐MB‐231 cell metabolism and promotes proliferation and migration. Since adipocytes are FA‐rich, and these FAs are transferred to BrCa cells, we sought to elucidate the FA metabolism of BrCa cells and their response to FA‐rich environments. MCF‐7 and MDA‐MB‐231 cells incubated in serum‐containing media supplemented with FAs accumulate extracellular FAs as intracellular triacylglycerols (TAG) in a dose‐dependent manner, with MDA‐MB‐231 cells accumulating more TAG. The differences in TAG levels were a consequence of distinct differences in intracellular partitioning of FAs, and not due to differences in the rate of FA uptake. Specifically, MCF‐7 cells preferentially partition FAs into mitochondrial oxidation, whereas MDA‐MB‐231 cells partition FAs into TAG synthesis. These differences in intracellular FA handling underpin differences in the sensitivity to palmitate‐induced lipotoxicity, with MDA‐MB‐231 cells being highly sensitive, whereas MCF‐7 cells are partially protected. The attenuation of palmitate‐induced lipotoxicity in MCF‐7 cells was reversed by inhibition of FA oxidation. Pretreatment of MDA‐MB‐231 cells with FAs increased TAG synthesis and reduced palmitate‐induced apoptosis. Our results provide novel insight into the potential influences of obesity on BrCa biology, highlighting distinct differences in FA metabolism in MCF‐7 and MDA‐MB‐231 cells and how lipid‐rich environments modulate these effects.

Lipids are known to influence tumour growth, inflammation and chemoresistance. However, the association of circulating lipids with the clinical outcome of metastatic castration-resistant prostate cancer (CRPC) is unknown. We investigated associations between the plasma lipidome and clinical outcome in CRPC. Lipidomic profiling by liquid chromatography-tandem mass spectrometry was performed on plasma samples from a Phase 1 discovery cohort of 96 CRPC patients. Results were validated in an independent Phase 2 cohort of 63 CRPC patients. Unsupervised analysis of lipidomic profiles (323 lipid species) classified the Phase 1 cohort into two patient subgroups with significant survival differences (HR 2.31, 95% CI 1.44-3.68, p = 0.0005). The levels of 46 lipids were individually prognostic and were predominantly sphingolipids with higher levels associated with poor prognosis. A prognostic three-lipid signature was derived (ceramide d18:1/24:1, sphingomyelin d18:2/16:0, phosphatidylcholine 16:0/16:0) and was also associated with shorter survival in the Phase 2 cohort (HR 4.8, 95% CI 2.06-11.1, p = 0.0003). The signature was an independent prognostic factor when modelled with clinicopathological factors or metabolic characteristics. The association of plasma lipids with CRPC prognosis suggests a possible role of these lipids in disease progression. Further research is required to determine if therapeutic modulation of the levels of these lipids by targeting their metabolic pathways may improve patient outcome.

Fatty acid β-oxidation (FAO) is the main bioenergetic pathway in prostate cancer (PCa) and a promising novel therapeutic vulnerability. Here we demonstrate therapeutic efficacy of targeting FAO in clinical prostate tumors cultured ex vivo, and identify DECR1, which encodes the rate-limiting enzyme for oxidation of polyunsaturated fatty acids (PUFAs), as robustly overexpressed in PCa tissues and associated with shorter relapse-free survival. DECR1 is a negatively-regulated androgen receptor (AR) target gene and, therefore, may promote PCa cell survival and resistance to AR targeting therapeutics. DECR1 knockdown in PCa cells selectively inhibited β-oxidation of PUFAs, inhibited proliferation and migration of PCa cells, including treatment resistant lines, and suppressed tumor cell proliferation in vivo. Mechanistically, targeting of DECR1 caused cellular accumulation of linoleic acid, enhanced mitochondrial oxidative stress and lipid peroxidation, and ferroptosis. These findings implicate PUFA oxidation via DECR1 as a previously unexplored facet of FAO that promotes survival of PCa cells.

Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are emerging as leading causes of liver disease worldwide and have been recognized as one of the major unmet medical needs of the 21st century. Our recent translational studies in mouse models, human cell lines, and well-characterized patient cohorts have identified serine/threonine kinase (STK)25 as a protein that coats intrahepatocellular lipid droplets (LDs) and critically regulates liver lipid homeostasis and progression of NAFLD/NASH. Here, we studied the mechanism-of-action of STK25 in steatotic liver by relative quantification of the hepatic LD-associated phosphoproteome from high-fat diet-fed Stk25 knockout mice compared with their wild-type littermates. We observed a total of 131 proteins and 60 phosphoproteins that were differentially represented in STK25-deficient livers. Most notably, a number of proteins involved in peroxisomal function, ubiquitination-mediated proteolysis, and antioxidant defense were coordinately regulated in Stk25(−/−) versus wild-type livers. We confirmed attenuated peroxisomal biogenesis and protection against oxidative and ER stress in STK25-deficient human liver cells, demonstrating the hepatocyte-autonomous manner of STK25’s action. In summary, our results suggest that regulation of peroxisomal function and metabolic stress response may be important molecular mechanisms by which STK25 controls the development and progression of NAFLD/NASH.

Annexin A6 (AnxA6) is a lipid-binding protein highly expressed in the liver, regulating cholesterol homeostasis and signaling pathways with a role in liver physiology. Here, we analyzed whether hepatic AnxA6 levels are affected by pathological conditions that are associated with liver dysfunction and liver injury. AnxA6 levels in the fatty liver of mice fed a high-fat diet, in ob/ob and db/db animals and in human fatty liver are comparable to controls. Similarly, AnxA6 levels appear unaffected in murine nonalcoholic steatohepatitis and human liver fibrosis. Accordingly, adiponectin, lysophosphatidylcholine, palmitate, and TGFbeta, all of which have a role in liver injury, do not affect AnxA6 expression in human hepatocytes. Likewise, adiponectin and IL8 do not alter AnxA6 levels in primary human hepatic stellate cells. However, in hepatic tumors of 18 patients, AnxA6 protein levels are substantially reduced compared to nontumorous tissues. AnxA6 mRNA is even increased in the tumors suggesting that posttranscriptional mechanisms are involved herein. Lipidomic analysis shows trends toward elevated cholesteryl ester and sphingomyelin in the tumor samples, yet the ratio of tumor to nontumorous AnxA6 does not correlate with these lipids. The current study shows that AnxA6 is specifically reduced in human hepatocellular carcinoma suggesting a role of this protein in hepatocarcinogenesis.

Prostate tumours are highly reliant on lipids for growth and survival. Emerging evidence suggests that the androgen receptor (AR) is a major regulator of lipid metabolism in prostate cancer (PCa), but the molecular mechanisms underlying such regulation are largely unknown. By integrating cistromic and transcriptomic data, we identified Acyl-CoA Synthetase Medium Chain Family Members 1 and 3 (ACSM1 and ACSM3) as putative new AR-regulated genes in prostate cancer. These factors are thought to function to activate fatty acids for their utilisation in energy production via mitochondrial beta-oxidation. AR regulation was validated by demonstrating modulation of ACSM1 and ACSM3 by androgen and anti-androgen treatment and confirming direct binding of AR to proximal cis-regulatory elements by chromatin immunoprecipitation (ChIP). ACSM1 and ACSM3 are upregulated in prostate tumours compared to non-malignant prostate tissues and are expressed more highly in prostate cancer than other cancer types. We subsequently applied metabolomics, lipidomics and functional assays to decipher the roles of ACSM1 and ACSM3 in prostate cancer cells. Knockdown of ACSM1 and ACSM3 in PCa cells resulted in growth arrest and ATP depletion, supporting a key role for both factors in energy production from fatty acids. Furthermore, lipidomic analysis of cells showed that poly-unsaturated fatty acids accumulate in response to loss of ACSM1 and ACSM3. Metabolomics revealed that cells adapt to loss of ACSM1 and ACSM3 by switching to a glycolytic phenotype. The metabolic dysregulation induced by knockdown of ACSM1 and ACSM3 caused mitochondrial oxidative stress and subsequent lipid peroxidation, eventually resulting in cell death. Accumulation of mitochondrial reactive oxygen species was abrogated by ferrostatin-1 (an iron chelator), suggesting that cell death was due to an iron-dependent form of apoptosis termed ferroptosis. Supporting this concept, over-expression of ACSM1 and ACSM3 elicited resistance to the ferroptosis inducers Erastin and ML210. Our study has revealed a novel mechanism by which AR regulates lipid metabolism in prostate cancer cells. Importantly, the critical role of ACSM1 and ACSM3 as key regulators of growth and protectors against ferroptosis emphasises their potential as novel therapeutic targets Citation Format: Raj Kumar Shrestha, Scott Townley, Adrienne Hanson, Marie Pickering, Zeyad D. Nassar, Chui Yan Mah, Mohammadreza A. Ghodsi, Andrew J. Hoy, Lake-Ee Quek, Wayne D. Tilley, Lisa M. Butler, Luke A. Selth. ACSM1 and ACSM3 regulate fatty acid oxidation in prostate cancer to promote growth and protect against oxidative stress [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-036.

Introduction: Fatty acid oxidation (FAO) is the dominant bioenergetic pathway in prostate cancer (PCa). Previous studies have demonstrated efficacy in targeting key FAO enzymes using in vitro and in vivo models of PCa. However, clinical evidence supporting FAO targeting in PCa is lacking. A recent study reported that FAO is upregulated in PCa cells and is essential for cell viability. Here, we demonstrated therapeutic efficacy of targeting FAO in clinical prostate tumors and identified DECR1 as a potential FAO-related survival factor. Methods and Results: To examine the therapeutic efficacy of targeting FAO in a more clinically-relevant setting, patient-derived prostate explants (PDE) were treated with etomoxir (FAO inhibitor). Results showed significant inhibition of cell proliferation (evaluated using Ki67 immunohistochemistry staining) in drug-treated PDE by a mean of 48% (n=13, p Conclusion: This study demonstrated for the first time clinically-relevant evidence that targeting FAO is efficacious and provides evidence for the importance of FAO, and specifically PUFA oxidation, in PCa progression. Importantly, this study identified DECR1 as a promising and novel therapeutic target for PCa. Citation Format: Chui Yan Mah, Zeyad D. Nassar, Ingrid J. Burvenich, Swati Irani, Margaret M. Centenera, Max Moldovan, David J. Lynn, Andrew J. Hoy, Johannes V. Swinnen, Lisa M. Butler. DECR1: The rate limiting enzyme of polyunsaturated fatty acid metabolism and a novel therapeutic target in prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 237.

The liver is a critical tissue for maintaining glucose, fatty acid, and cholesterol homeostasis. Primary hepatocytes represent the gold standard for studying the mechanisms controlling hepatic glucose, lipid, and cholesterol metabolism in vitro. However, access to primary hepatocytes can be limiting, and therefore, other immortalized hepatocyte models are commonly used. Here, we describe substrate metabolism of cultured AML12, IHH, and PH5CH8 cells, hepatocellular carcinoma-derived HepG2s, and primary mouse hepatocytes (PMH) to identify which of these cell lines most accurately phenocopy PMH basal and insulin-stimulated metabolism. Insulin-stimulated glucose metabolism in PH5CH8 cells, and to a lesser extent AML12 cells, responded most similarly to PMH. Notably, glucose incorporation in HepG2 cells were 14-fold greater than PMH. The differences in glucose metabolic activity were not explained by differential protein expression of key regulators of these pathways, for example glycogen synthase and glycogen content. In contrast, fatty acid metabolism in IHH cells was the closest to PMHs, yet insulin-responsive fatty acid metabolism in AML12 and HepG2 cells was most similar to PMH. Finally, incorporation of acetate into intracellular-free cholesterol was comparable for all cells to PMH; however, insulin-stimulated glucose conversion into lipids and the incorporation of acetate into intracellular cholesterol esters were strikingly different between PMHs and all tested cell lines. In general, AML12 cells most closely phenocopied PMH in vitro energy metabolism. However, the cell line most representative of PMHs differed depending on the mode of metabolism being investigated, and so careful consideration is needed in model selection.

Glutamine is conditionally essential in cancer cells, being utilized as a carbon and nitrogen source for macromolecule production, as well as for anaplerotic reactions fuelling the tricarboxylic acid (TCA) cycle. In this study, we demonstrated that the glutamine transporter ASCT2 (SLC1A5) is highly expressed in prostate cancer patient samples. Using LNCaP and PC‐3 prostate cancer cell lines, we showed that chemical or shRNA‐mediated inhibition of ASCT2 function in vitro decreases glutamine uptake, cell cycle progression through E2F transcription factors, mTORC1 pathway activation and cell growth. Chemical inhibition also reduces basal oxygen consumption and fatty acid synthesis, showing that downstream metabolic function is reliant on ASCT2‐mediated glutamine uptake. Furthermore, shRNA knockdown of ASCT2 in PC‐3 cell xenografts significantly inhibits tumour growth and metastasis in vivo, associated with the down‐regulation of E2F cell cycle pathway proteins. In conclusion, ASCT2‐mediated glutamine uptake is essential for multiple pathways regulating the cell cycle and cell growth, and is therefore a putative therapeutic target in prostate cancer. © 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Emerging data have linked certain features of clinical prostate cancer (PCa) to obesity and, more specifically, increased adiposity. Whereas the large number of clinical studies and meta-analyses that have explored the associations between PCa and obesity have shown considerable variability, particularly in relation to prostate cancer risk, there is an accumulating weight of evidence consistently linking obesity to greater aggressiveness of disease. In probing this association mechanistically, it has been posited that peri-prostatic adipose tissue (PPAT), a significant component of the prostate microenvironment, may be a critical source of fatty acids and other mitogens and thereby influences PCa pathogenesis and progression. Notably, several recent studies have identified secreted factors from both PPAT and PCa that potentially mediate the two-way communication between these intimately linked tissues. In the present review, we summarize the available literature regarding the relationship between PPAT and PCa, including the potential biological mediators of that relationship, and explore emerging areas of interest for future research endeavours.

Metabolic reprogramming represents an important hallmark of cancer cells. Besides de novo fatty acid synthesis, it is now clear that cancer cells can acquire Fatty Acids (FA) from tumor-surrounding adipocytes to increase their invasive capacities. Indeed, adipocytes release FA in response to tumor secreted factors that are transferred to tumor cells to be either stored as triglycerides and other complex lipids or oxidized in mitochondria. Like all cells, FA can be released over time from triglyceride stores through lipolysis and then oxidized in mitochondria in cancer cells. This metabolic interaction results in specific metabolic remodeling in cancer cells, and underpins adipocyte stimulated tumor progression. Lipolysis and fatty acid oxidation therefore represent novel targets of interest in the treatment of cancer. In this review, we summarize the recent advances in our understanding of the metabolic reprogramming induced by adipocytes, with a focus on breast cancer. Then, we recapitulate recent reports studying the effect of lipolysis and fatty acid oxidation inhibitors on tumor cells and discuss the interest to target these metabolic pathways as new therapeutic approaches for cancer.

Annexin A6 (AnxA6) has been implicated in the regulation of endo-/exocytic pathways, cholesterol transport, and the formation of multifactorial signaling complexes in many different cell types. More recently, AnxA6 has also been linked to triglyceride storage in adipocytes. Here we investigated the potential role of AnxA6 in fatty acid (FA) - induced lipid droplet (LD) formation in hepatocytes. AnxA6 was associated with LD from rat liver and HuH7 hepatocytes. In oleic acid (OA) -loaded HuH7 cells, substantial amounts of AnxA6 bound to LD in a Ca2+-independent manner. Remarkably, stable or transient AnxA6 overexpression in HuH7 cells led to elevated LD numbers/size and neutral lipid staining under control conditions as well as after OA loading compared to controls. In contrast, overexpression of AnxA1, AnxA2 and AnxA8 did not impact on OA-induced lipid accumulation. On the other hand, incubation of AnxA6-depleted HuH7 cells or primary hepatocytes from AnxA6 KO-mice with OA led to reduced FA accumulation and LD numbers. Furthermore, morphological analysis of liver sections from A6-KO mice revealed significantly lower LD numbers compared to wildtype animals. Interestingly, pharmacological inhibition of cytoplasmic phospholipase A2α (cPLA2α)-dependent LD formation was ineffective in AnxA6-depleted HuH7 cells. We conclude that cPLA2α-dependent pathways contribute to the novel regulatory role of hepatic AnxA6 in LD formation.

Although a nonessential amino acid in normal cells, the demand for glutamine is dramatically increased throughout malignant transformation to support increased metabolic demands; namely, provision of catabolic substrates for ATP production and anabolic substrates for the citric acid cycle and subsequent macromolecule biosynthesis, as well as potentiating the uptake of other critical amino acids by acting as an obligate exchange substrate. Elevated expression of glutamine metabolism-related genes, MYC-driven transcriptional events, and increased consumption and reliance on glutamine are all associated with aggressive breast cancers, including the high-risk triple-negative (TN) subtype. We recently showed that in breast cancer cells, glutamine uptake by the small neutral amino acid transporter, ASCT2, is required to sustain TN cell growth in vitro and in vivo. We therefore hypothesised that highly proliferative TN breast cancers that are sensitive to ASCT2 inhibition may have unique metabolic signatures that could be additionally exploited for therapeutic purposes. Using radiolabelled and stable isotope tracing methods, we analysed in detail glutamine catabolism and its conversion into various metabolic products such as tricarboxylic acid (TCA) cycle intermediates, lipids, nucleotides, and amino acids in human breast cancer cell lines in vitro and ex vivo. These analyses revealed distinct metabolic effects across cell lines representing different subtypes of breast cancer, both at baseline and when glutamine uptake was blocked in vitro by L-gamma-glutamyl-p-nitroanilide (GPNA), a pharmacological inhibitor of ASCT2. These data confirm a broad reliance on glutamine availability in TN breast cancers, where glutamine anapleurosis was significantly greater than in the Luminal A subtype. Analysis of gene expression data from the METABRIC and TCGA patient datasets and the CCLE cell line dataset showed a specific upregulation of multiple glutamine metabolism enzymes in TN breast cancer patient samples and cell lines compared to the Luminal A subtype. These data suggest a rewiring of glutamine metabolism in TN breast cancer that utilises their addiction to glutamine to fuel cell growth. Furthermore, these data provide an additional suite of glutamine metabolic enzymes that may be therapeutically targeted in the hard-to-treat TN breast cancer subset. Citation Format: Michelle van Geldermalsen, Lake-Ee Quek, Nigel Turner, Seher Balaban, Andrew Hoy, Angel Pang, Yi Fang Guan, Kanu Wahi, Qian Wang, Jeff Holst. Vulnerabilities in glutamine metabolism define triple-negative from Luminal A breast cancer subsets [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-02-09.

Vitamin D status, measured as serum 25-hydroxyvitamin D (25OHD) concentration, is determined by rates of input and of degradation. The half-life of 25OHD is surprisingly long for a steroid and much longer than its blood transporter, vitamin D binding protein. There is some evidence to suggest that vitamin D itself is stored in fat, whereas 25OHD concentrations are usually related to muscle-related parameters such as lean body mass and exercise. Both muscle and fat cells come from the mesenchymal cell lineage. We recently published evidence for net uptake of 25OHD into differentiated muscle cells, in a process that was megalin dependent, and speculated that this uptake might contribute to the extended half-life of 25OHD. Whether 25OHD is also taken up into cells of the adipocyte lineage is not clear. In the current study, we used the C2 muscle cell line as a source of myoblasts that were differentiated in culture to myotubes and 3T3-L1 pre-adipocytes that were differentiated into adipocytes in culture. We incubated the cells with trititated 25OHD and measured net uptake 4 and 16h afterwards. Differentiated myotubes took up labeled 25OHD in a time-dependent process to a far greater extent than myoblasts. In contrast, pre-adipocytes, but not differentiated adipocytes, accumulated labeled 25OHD in a time-dependent manner, though to a lesser extent than myotubes. Myotubes, but not myoblasts, showed megalin expression by immunohistochemistry. Pre-adipocytes, but not adipocytes, also showed expression of megalin. Since skeletal muscle consists mainly of differentiated muscle cells, while adipose tissue is mainly differentiated fat cells, it seems likely that muscle, but not fat tissue, provides a large extravascular pool through which 25OHD circulates and that this protects 25OHD from degradation. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

Objective Oxygen delivery, underpinned by vascular tone, is the principle limiting factor in the study of skeletal muscle physiology, particularly during muscle contraction. The aim of this study was to develop an autologous perfused rat hind limb preparation for the study of skeletal muscle contractile function. Methods Adult Wistar rats were surgically prepared using a by-pass system for pump-controlled arterial blood flow to, and venous return from the hind limb during periods of quiescence and twitch contraction of the gastrocnemius-plantaris-soleus muscle bundle. Results During rest, hind limb perfusion pressure (102 ± 5 mmHg) was not different to systemic arterial pressure (99 ± 4 mmHg). Hind limb pressure was responsive to vasoconstrictors and vasodilators (±50 mmHg). The arterial PO2 (100 ± 3 mmHg), O2 saturation, and acid–base balance (pH: 7.42 ± 0.01) contributed to resting hind limb (a-v)O2 difference (4.8 ± 0.5 mL/100 mL) and VO2 (0.31 ± 0.03 μmol/g/min wet weight). Repetitive isometric twitch tension (1 Hz, 0.05 ms, 10 minutes) was best maintained at a flow rate of 2 mL/min (VO2 increased fivefold during muscle contraction) and efficiency of oxygen use increased from 0.27 ± 0.08–0.52 ± 0.07 N/μmol/min. Conclusion The autologous rat hind limb provided resting vascular tone allowing maintenance of perfusion pressure at flows within the physiological range. Oxygen delivery supported repetitive twitch contractions and facilitated measurement of active metabolism.

Background Pancreatic cancer has a five-year survival rate of ~8%, with characteristic molecular heterogeneity and restricted treatment options. Targeting metabolism has emerged as a potentially effective therapeutic strategy for cancers such as pancreatic cancer, which are driven by genetic alterations that are not tractable drug targets. Although somatic mitochondrial genome (mtDNA) mutations have been observed in various tumors types, understanding of metabolic genotype-phenotype relationships is limited. Methods We deployed an integrated approach combining genomics, metabolomics, and phenotypic analysis on a unique cohort of patient-derived pancreatic cancer cell lines (PDCLs). Genome analysis was performed via targeted sequencing of the mitochondrial genome (mtDNA) and nuclear genes encoding mitochondrial components and metabolic genes. Phenotypic characterization of PDCLs included measurement of cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using a Seahorse XF extracellular flux analyser, targeted metabolomics and pathway profiling, and radiolabelled glutamine tracing. Results We identified 24 somatic mutations in the mtDNA of 12 patient-derived pancreatic cancer cell lines (PDCLs). A further 18 mutations were identified in a targeted study of ~1000 nuclear genes important for mitochondrial function and metabolism. Comparison with reference datasets indicated a strong selection bias for non-synonymous mutants with predicted functional effects. Phenotypic analysis showed metabolic changes consistent with mitochondrial dysfunction, including reduced oxygen consumption and increased glycolysis. Metabolomics and radiolabeled substrate tracing indicated the initiation of reductive glutamine metabolism and lipid synthesis in tumours. Conclusions The heterogeneous genomic landscape of pancreatic tumours may converge on a common metabolic phenotype, with individual tumours adapting to increased anabolic demands via different genetic mechanisms. Targeting resulting metabolic phenotypes may be a productive therapeutic strategy. Electronic supplementary material The online version of this article (doi:10.1186/s40170-017-0164-1) contains supplementary material, which is available to authorized users.