extracellular fatty acids are the major contributor to ... · 15-01-2019 · 48 extracellular fas...

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Extracellular Fatty Acids are the Major Contributor to Lipid Synthesis in Prostate Cancer 1

Seher Balaban 1*

, Zeyad D. Nassar 2,3*

, Alison Y. Zhang 4,5,6*

, Elham Hosseini-Beheshti 1, Margaret 2

M. Centenera 2,3

, Mark Schreuder 1,7

, Hui-Ming Lin 4, Atqiya Aishah

1, Bianca Varney

1, Frank Liu-3

Fu 1, Lisa S. Lee

1, Shilpa R. Nagarajan

1, Robert F. Shearer

4, Rae-Anne Hardie

5,8, Nikki L. 4

Raftopulos 1, Meghna S. Kakani

1, Darren N. Saunders

9, Jeff Holst

5,8, Lisa G. Horvath

4,5,6,9,10, Lisa 5

M. Butler 2,3

, and Andrew J. Hoy 1 #

6

1 Discipline of Physiology, School of Medical Sciences & Bosch Institute, Charles Perkins Centre, Faculty of Medicine 7

and Health, The University of Sydney, NSW 2006, Australia. 2 Adelaide Medical School and Freemasons Foundation 8

Centre for Men's Health, University of Adelaide, Adelaide, South Australia, 5005, Australia. 3 South Australian Health 9

and Medical Research Institute, Adelaide, South Australia, 5001, Australia. 4 Cancer Division, The Kinghorn Cancer 10

Centre/Garvan Institute for Medical Research, Darlinghurst, NSW, Australia. 5 Faculty of Medicine and Health, 11

University of Sydney, Sydney, New South Wales, Australia. 6 Chris O’Brien Lifehouse, Camperdown, NSW, Australia. 12 7 Faculty of Medicine, University of Utrecht, The Netherlands. 8 Origins of Cancer Program, Centenary Institute, 13

University of Sydney, Camperdown, New South Wales, Australia. 9 School of Medical Sciences, UNSW Australia, 14

Sydney, NSW 2052 Australia. 10 Royal Prince Alfred Hospital, Camperdown, NSW, Australia. 15

* these authors contributed equally 16

Abbreviated title: Fatty acid metabolism in prostate cancer 17

Keywords: fatty acid oxidation, apoptosis, prostate cancer, de novo lipogenesis, triglyceride 18

synthesis 19

Financial Support 20

LMB, AJH and JH acknowledge grant support from The Movember Foundation/Prostate Cancer 21

Foundation of Australia (MRTA3 and MRTA1). AJH is supported by a University of Sydney 22

Robinson Fellowship and was supported by Helen and Robert Ellis Postdoctoral Research 23

Fellowship from the Sydney Medical School Foundation and funding from the University of 24

Sydney. R-AH and AJH received support from the Sydney Medical School. SB was a recipient of a 25

University of Sydney Australian Postgraduate Award. ZDN is supported by an Early Career 26

Fellowship from the National Health and Medical Research Council of Australia and John Mills 27

Young Investigator Award from the Prostate Cancer Foundation of Australia. LMB is supported by 28

a Principal Cancer Research Fellowship produced with the financial and other support of Cancer 29

Council SA's Beat Cancer Project on behalf of its donors and the State Government of South 30

Australia through the Department of Health and was supported by a Future Fellowship from the 31

Australian Research Council (FT130101004). DNS was supported by the National Health and 32

Medical Research Council (project grant GNT1052963). MS was supported by funding from the 33

Dutch Cancer Institute KWF. 34

on October 10, 2020. © 2019 American Association for Cancer Research.mcr.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on January 15, 2019; DOI: 10.1158/1541-7786.MCR-18-0347

http://mcr.aacrjournals.org/

2

# Corresponding author: Andrew J. Hoy, Ph.D. 35

Discipline of Physiology, Charles Perkins Centre, University of Sydney, NSW, Australia 2006 36

Phone: +61 2 9351 2514 Email: [email protected] 37

Competing Interests 38

The authors declare no competing financial interests. 39

Word count: 6 312 words for main section 40

Total number of figures: 6 plus 3 supplementary figures 41



mailto:[email protected]


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Abstract: Prostate cancer cells exhibit altered cellular metabolism but, notably, not the hallmarks of 42

Warburg metabolism. Prostate cancer cells exhibit increased de novo synthesis of fatty acids (FA); 43

however, little is known about how extracellular FAs, such as those in the circulation, may support 44

prostate cancer progression. Here, we show that increasing FA availability increased intracellular 45

triacylglycerol content in cultured patient-derived tumor explants, LNCaP and C4-2B spheroids, a 46

range of prostate cancer cells (LNCaP, C4-2B, 22Rv1, PC-3), and prostate epithelial cells (PNT1). 47

Extracellular FAs are the major source (~83%) of carbons to the total lipid pool in all cell lines, 48

compared to glucose (~13%) and glutamine (~4%), and FA oxidation rates are greater in prostate 49

cancer cells compared to PNT1 cells, which preferentially partitioned extracellular FAs into 50

triacylglycerols. Due to the higher rates of FA oxidation in C4-2B cells, cells remained viable when 51

challenged by the addition of palmitate to culture media and inhibition of mitochondrial FA 52

oxidation sensitized C4-2B cells to palmitate-induced apoptosis. Whereas in PC-3 cells, palmitate 53

induced apoptosis, which was prevented by pre-treatment of PC-3 cells with FAs, and this 54

protective effect required DGAT-1-mediated triacylglycerol synthesis. These outcomes highlight 55

for the first-time heterogeneity of lipid metabolism in prostate cancer cells and the potential 56

influence that obesity-associated dyslipidemia or host circulating has on prostate cancer 57

progression. 58

Implications: Extracellular-derived FAs are primary building blocks for complex lipids and 59

heterogeneity in FA metabolism exists in prostate cancer that can influence tumor cell behavior. 60




4

INTRODUCTION 61

Metabolic changes during malignant transformation are recognized as a major hallmark of cancer 62

(1). In general, cancer cells exhibit increased glucose uptake and enhanced rates of glycolysis 63

resulting in lactate and energy production, termed the Warburg Effect (2). Other cancer-related 64

alterations in intracellular metabolic pathways include elevated synthesis of nucleotides, proteins 65

and fatty acids (FA) to support increased rates of growth and division (3). Significant attention has 66

centered on altered glucose and glutamine metabolism, including their roles as precursors for de 67

novo FA synthesis / lipogenesis (4-6), yet the contribution of FAs to cancer cell biology remains 68

elusive. FAs are the main structural components of biological membranes and are building blocks 69

for complex lipids such as triacylglycerols (TAG) and membrane phospholipids, and signaling 70

intermediates including diacylglycerol, phosphoinositols, sphingosine and phosphatidic acid (7). 71

The intracellular FA pool that acts as precursors for complex lipid synthesis is supplied by 72

extracellular sources such as lipoprotein-contained TAGs and adipose-derived free FAs, as well as 73

through de novo synthesis. Importantly, obese patients tend to have higher stage and a high 74

mortality rate from a range of cancers (see review (8)). These patients typically have increased 75

adiposity and dyslipidemia, resulting in a lipid-rich extratumoral environment. 76

Prostate cancer is the most common cancer in men and the second leading cause of male cancer-77

related death. The mainstay of treatment for advanced prostate cancer is androgen deprivation 78

therapy; however, this treatment is not curative, and patients inevitably develop a lethal form of the 79

disease termed castration-resistant prostate cancer (9). Unlike most other carcinomas, prostate 80

cancer is characterized by a slow glycolytic rate and may be more reliant on FA oxidation to 81

provide ATP for cell proliferation and growth, due to increased rates of citrate oxidation (10). 82

Prostate cancer also exhibits a number of lipid-specific features, including increased lipid droplet 83

number and size in high grade carcinomas compared to low grade prostate carcinomas and normal 84

prostate tissue (11), and enhanced rates of de novo FA synthesis (12-14). Whilst the functional 85

significance of these phenotypes is yet to be elucidated, they likely arise from aberrant activation of 86

SREBPs and enhanced expression of FASN (14-16). The resultant increase in de novo FA 87

synthesis, which uses acetyl-CoA as a major substrate, is the basis for evaluating 11

C-acetate-PET 88

for diagnosis and staging (17); however, the influence of factors other than tumor metabolism on 89

acetate uptake results in high false-positive results (17). One potential driving factor that influences 90

11C-acetate uptake and metabolism could be the extratumoral lipid environment. It is known that 91

high extracellular lipid levels influence glucose metabolism in type 2 diabetes (18) and in breast 92

cancer (19-21), and can directly influence the rate of de novo FA synthesis from non-lipid sources 93

(i.e. glucose- or glutamine-derived acetyl-CoA) (22). It has also been reported that dietary fat and 94




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metabolic disorders, including dyslipidemia and obesity are adverse prognostic factors influencing 95

disease behavior (see review (23)). Recently, we identified a prognostic three-lipid signature that is 96

associated with poor prognosis in men with lethal metastatic castration-resistant prostate cancer 97

(24). Collectively, these observations suggest that the extracellular lipid environment may influence 98

prostate cancer FA metabolism and behavior, yet this relationship remains to be characterized. 99

The aim of this study was to assess FA metabolism in patient-derived explants and in a range of 100

prostate cancer cell lines, and elucidate the role of FA metabolism in prostate cancer cell survival. 101

Insights into these situations may provide a greater understanding into the potential role that 102

elevated extracellular FA levels may play in obesity-induced prostate cancer progression (see 103

reviews (23,25)). 104




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MATERIALS AND METHODS 105

Cell Culture 106

The human prostate epithelial cell line PNT1 and the human prostate carcinoma cell lines LNCaP 107

(androgen receptor positive, androgen sensitive), C4-2B, 22Rv1 (androgen receptor positive, 108

androgen independent), PC-3 (androgen receptor negative, androgen resistant) were obtained from 109

the American Type Culture Collection (ATCC). Cell lines are validated annually by Garvan 110

Molecular Genetics using a test based on the Powerplex 18D kit (DC1808, Promega) and tested for 111

mycoplasma every 3 months (MycoAlert™ mycoplasma detection kit, Lonza). All cell lines were 112

cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Life Technologies Australia 113

Pty Ltd., Scoresby VIC, Australia) supplemented with 10% fetal calf serum (FCS; HyClone, GE 114

Healthcare Life Sciences, USA) and 100 IU/ml penicillin and 100 IU/ml streptomycin (Life 115

Technologies Australia Pty Ltd., Scoresby VIC, Australia). The passage numbers of all cell lines 116

were below 20 between thawing and use in the experiments. 117

To create the spheroids, approximately 1 x 106 cells were obtained by trypsinization from growing 118

monolayer cultures. Cells (2 x 104 /well) were seeded in 96-well, ultra-low attachment, round-119

bottom plates (Costar, Corning). These cells were then centrifuged at 1500×g for 10 min and 120

incubated at 37°C in a humidified 5% CO2 incubator for 7 days. The cells were kept without 121

agitation except when fresh growth medium was administered every 72 hours. 122

To inhibit DGAT-1 activity, cells were treated with 60 nM of AZD3988 (Tocris Bioscience, 123

Invitrogen) (26) for 24 h in RPMI, 10 % FCS and no antibiotics. After treatment, cells were washed 124

and sensitivity to palmitate-induced apoptosis or cell growth in fresh RPMI containing 10 % FCS 125

was assessed. To inhibit FA oxidation, cells were treated with 100 µM etomoxir (Sigma) in RPMI, 126

10 % FCS and no antibiotics. 127

Cell Transfection 128

Cells were seeded two days before the experiment and transfected using RNAiMAX transfection 129

reagent (#13778075; Thermo Fisher Scientific) and 25 pmol Pooled CPT1A siRNA (ON-130

TARGETplus SMARTpool L-009749-00-0005; Thermo Scientific, Chicago, IL) (27) or ON-131

TARGETplus Non-targeting Pool (D-001810-10-05) according to the manufacturer's instructions. 132

After 48 hours, cells were washed and sensitivity to palmitate-induced apoptosis and fatty acid 133

oxidation assessed. 134

Patient-Derived Explants 135




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Human ethical approval for this project was obtained from the University of Adelaide Human 136

Research Ethics Committee, St Andrew’s Hospital Research Ethics Committee (reference number 137

80; Adelaide, South Australia) or St Vincent’s Hospital’s Human Research Ethics Committee 138

(12/231; Sydney, New South Wales). Fresh prostate cancer specimens were obtained with written 139

informed consent through the Australian Prostate Cancer BioResource from men undergoing 140

robotic radical prostatectomy at either the St Andrew’s Hospital (Adelaide, South Australia) or St 141

Vincent’s Clinic (Sydney, New South Wales). Patient-derived explants (PDEs) were prepared and 142

cultured as previously reported (28,29). Briefly, a 6 mm core of tissue was dissected into 1 mm3 143

pieces and cultured in triplicate on a pre-soaked gelatin sponge (Johnson and Johnson, New 144

Brunswick, NJ) in 24-well plates containing RPMI 1640 with 10 % FBS, antibiotic/antimycotic 145

solution (Sigma, St Louis, MO), 0.01 mg/ml hydrocortisone, and 0.01 mg/ml insulin (Sigma). PDEs 146

were cultured at 37°C for up to 72 h and then rinsed twice in ice cold PBS prior to being snap 147

frozen. 148

Lipid-loading of Cells, Spheroids and PDEs 149

Cell lines, spheroids and PDEs were incubated in RPMI medium supplemented with differing 150

concentrations of either oleate only, or 1:2:1 palmitate:oleate:linoleate (FA Mix; Sigma) as 151

indicated plus 10% FCS (wt/vol.) and no antibiotics for 24 h. 152

Substrate Metabolism 153

PDE fatty acid uptake: explants were cultured in assay media containing 2% fatty acid-free BSA, 154

0.2 mmol/l cold oleate (Sigma), and 0.2 µCi/ml [1-14

C]oleate (Perkin Elmer, Boston, MA) in the 155

presence or absence of 100 nM insulin. Snap frozen tissues were homogenized in 100 µL PBS using 156

a Precellys24 tissue homogenizer (Bertin Technologies, France) and lysate transferred to 900 µL 157

Ultima Gold scintillation fluid for counting on a Tri-Carb® 2810TR liquid scintillation analyzer 158

(Perkin Elmer). 159

Extracellular-derived FA metabolism: cells were maintained in FCS-containing media prior to 160

experimentation. Cells were washed in warmed PBS, then incubated in assay medium containing 161

0.5 mmol/l cold oleate or palmitate, [1-14

C]oleate or [1-14

C]palmitate (0.5 μCi/ml; PerkinElmer, 162

Boston, MA) conjugated to 2% (wt/vol.) FA-free BSA and 1 mM L-carnitine in low glucose 163

DMEM for 4 h. Mitochondrial oxidation was determined from 14

CO2 production as previously 164

described (30). Cells were harvested on ice-cold PBS to determine 14

C-oleate incorporation into 165

intracellular lipid pools and protein content. FA uptake was calculated as the sum of 14

CO2, 14

C 166

activity in the aqueous phase and 14

C incorporation into lipid containing organic phase of cell 167

lysates. 168




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Intracellular (TAG)-derived FA metabolism: cells were maintained in FCS-containing media prior 169

to experimentation. Cells were washed in warmed PBS, then pulsed overnight for 18 h in assay 170

media containing 2% fatty acid-free BSA, with [1-14

C]oleate (1 μCi/ml; PerkinElmer, Boston, MA) 171

and cold oleate (C4-2B: 20 or 150 µM; PC-3: 80 or 300 µM) to pre-label the endogenous TAG 172

pool. Following the pulse, the specific activity of the TAG pool was determined in a cohort of cells 173

by measuring the 14

C activity in the TAG following lipid extraction and TLC as well as the 174

biochemical assessment of the TAG pool (see Biochemical Measures). TAG-derived FA oxidation 175

(endogenous FA oxidation) was determined by measuring 14

CO2 production in another cohort run in 176

parallel where cells were chased for 4 h in RPMI media containing 0.5% FA-free BSA and 1 mM L-177

carnitine. 178

Glucose and glutamine metabolism: cells were maintained in FCS-containing media prior to 179

experimentation. Cells were washed in warmed PBS, then incubated in the same media for oleate 180

metabolism but with the either U-[14

C]-D-Glucose or 1-[14

C]-L-Glutamine (0.5µCi/ml, Perkin 181

Elmer Inc., USA) in place of [1-14

C]oleate and placed on cells for 4 h. Substrate uptake was 182

calculated as the sum of 14

CO2, 14

C activity in the aqueous phase and 14

C incorporation into lipid 183

containing organic phase of cell lysates. 184

Cellular lipids were extracted using the Folch method (31). Lipids were separated by TLC using 185

heptane-isopropyl ether-acetic acid (60:40:3, v/v/v) as developing solvent for TAG and 186

phospholipids or by a two-step solvent system for ceramides where TLC plates were developed to 187

one-third of the total length of the plate in chloroform: methanol: 25 % NH3 (20:4:0.2, v/v/v), dried, 188

then rechromotographed in heptane: isopropyl ether: acetic acid (60:40:3, v/v/v). 14

C activity in the 189

TAG, phospholipid and ceramide bands was determined by scintillation counting. 190

The contribution of oleate, glucose and glutamine to lipid synthesis was calculated by summing the 191

14C activity from the organic phase following lipid extraction for each substrate, expressed as 192

pmol/min/mg, then calculating the percent contribution for each substrate. 193

Biochemical Measures 194

Monolayer cultured cell and spheroid TAGs were extracted using the method of Folch et al (31) and 195

quantified using an enzymatic colorimetric method (GPO-PAP reagent, Roche Diagnostics). Cell 196

protein content was determined using Pierce Micro BCA protein assay (Life Technologies Australia 197

Pty Ltd., Scoresby VIC, Australia). 198

Visualization of Lipid Droplets 199

PDEs were washed twice in ice cold PBS and snap frozen. Frozen sections of 5 µm thickness were 200




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dried then washed with propylene glycol prior to staining with warm (60°C) Oil Red O for 10 mins. 201

Tissues were differentiated in 85% propylene glycol, rinsed twice in distilled water and 202

counterstained with hematoxylin for 30 secs. Tissues were rinsed in distilled water and mounted 203

with aqueous mounting medium. Stained tissues were visualized using a Panoramic 250 Digital 204

Slide Scanner (3D Histech, Budapest, Hungary) and staining quantitated using ImageJ software 205

(v1.49t). 206

Spheroids were washed in PBS, fixed with 4% PFA. Spheroids were then washed with 60% 207

Isopropanol and stained with Oil Red O for 15 minutes at room temperature. Spheroids were 208

washed with Isopropanol, distilled water, and then embedded in OCT, before cryosectioning and 209

counterstained with hematoxylin. The stained spheroid droplets were observed using a Leica 210

DM4000 microscope. 211

PDE Immunohistochemistry 212

ATGL staining was performed on PDEs from 10 patients on the Leica BOND RX platform. 213

Sections of 4 µm thick paraffin-embedded blocks were dewaxed and rehydrated with Bond wash 214

solution (Ref: AR9222). The slides underwent antigen retrieval using a standardized heat-induced 215

epitope retrieval protocol (HIER 20 minutes with ER2). The primary antibody against ATGL 216

(#2138S, Cell Signaling, MA, USA) was applied and incubated for 15 minutes, followed by 217

application of a post primary mouse antibody for 8 minutes, followed by a secondary rabbit 218

antibody for 8 minutes (both part of Leica Bond Polymer Refine Detection system). Bound 219

antibody was stained with 3, 3-diaminobenzidine tetrahydrochloride (Mixed DAB Refine) and then 220

counterstained with hematoxylin. Optimal primary antibody concentration was determined by serial 221

dilutions, optimizing for maximal signal without background interference. The final ATGL 222

antibody dilution used was 1:500. ATGL staining was quantified only in areas of tumor as 223

determined by Clinical Pathologist. Only 5 of the 10 PDEs that were sectioned and stained for 224

ATGL contained tumor. 225

Western Blot Analysis 226

Protein extraction from mono-cultures was performed as described previously (32). Cell lysates 227

were subjected to SDS-PAGE, transferred to PVDF membranes (Merck Millipore), and then 228

immunoblotted with antibodies for anti-ATGL (2138S), anti-PARP (9532S), anti-ATF4 (11815S) 229

and anti-GAPDH (2118S) obtained from Cell Signaling Technology (Danvers, MA), anti-CPT1A 230

(ab128568), anti-DGAT-1 (ab54037), and anti-DGAT-2 (ab59493) from Abcam (Cambridge, MA, 231

USA). Chemiluminescence performed using Luminata Crescendo Western HRP Substrate (Merck 232




10

Millipore) and imaged using the Bio-Rad ChemiDoc MP Imaging System (Bio-Rad laboratories, 233

Hercules, CA, USA) using Image Lab software 4.1 (Bio-Rad laboratories, Hercules, CA, USA). 234

Palmitate Treatment and Cell Viability 235

PC-3 and C4-2B cells were plated in triplicate in 96 well plates (3x103 cells/ well) and a group of 236

cells were then lipid-loaded for 24h, with ethanol used as a vehicle control. The following day, the 237

media was removed, cells were washed and fresh RPMI media containing 10 % FCS supplemented 238

with 250 μM palmitate (Sigma-Aldrich, NSW, AU) or ethanol as a vehicle control added. In 239

separate experiments, a palmitate dose response was performed using fresh RPMI media containing 240

10 % FCS supplemented with 62.5, 125, 250 or 500 μM palmitate or ethanol as a vehicle control. 241

At defined time points stated in figure legends, MTT assays were performed as described 242

previously (33), cells counted and viability assessed by trypan blue dye exclusion at indicated time 243

points. In another cohort, cells were lysed for protein content determination after 4 days of 244

treatment or immunoblot analysis after 24h of palmitate treatment. 245

Statistical Analysis 246

Statistical analyses were performed with Graphpad Prism 7.03 (Graphpad Software, San Diego, 247

CA). Differences among groups were assessed with appropriate statistical tests noted in figure 248

legends. P ≤ 0.05 was considered significant. Data are reported as mean ± SEM of at least 3 249

independent determinations. 250




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RESULTS 251

Increasing FA availability increases triacylglycerol content in patient-derived explants and a 252

range of human prostate cancer cell lines 253

Firstly, we assessed the influence of the extracellular lipid environment on neutral lipid levels in 254

clinical prostate tumors cultured as patient-derived explants (PDEs). PDEs incubated in FCS-255

containing media supplemented with 500 µM of the monounsaturated FA oleate for 72 h displayed 256

increased intracellular neutral lipid levels (as determined by Oil Red-O staining; Figure 1A), and a 257

trend for increased protein levels (P=0.09) of the lipid droplet lipase adipose triglyceride lipase 258

(ATGL; Figure 1B & C). PDEs also accumulated radiolabeled oleate in a time dependent manner 259

(Figure 1D). FA uptake is stimulated by insulin in many tissues (34) and here this was also evident 260

in PDEs (Figure 1D). Collectively, these data demonstrated that castrate-sensitive prostate cancer 261

was sensitive to the extracellular lipid environment and accumulates FAs as neutral lipids. 262

We further explored these observations using 3D spheroid models to assess the time and dose-263

dependent effects of extracellular FAs on neutral lipid levels. Incubating androgen-sensitive LNCaP 264

spheroids in 500 µM oleate increased Oil Red-O staining, with stronger staining observed following 265

72 h of culture (Figure 1E). Increased Oil Red-O staining in LNCaP spheroids was associated with 266

an increase in the neutral lipid TAG in a dose and time-dependent manner (Figure 1F). Similar 267

patterns were observed in androgen-insensitive C4-2B spheroids (Figure 1G & H). 268

We next assessed the effects of the extracellular lipid environment on intracellular TAG levels in a 269

range of prostate cancer cell lines and normal prostate epithelial cells. Non-malignant prostate 270

epithelial PNT1 cells (Figure 1I), AR positive and androgen sensitive LNCaP cells, AR positive and 271

androgen independent C4-2B and 22Rv1 cells (Figure 1J-L), and AR negative PC-3 cells (Figure 272

1M) accumulated intracellular TAG in a dose-dependent manner when cultured in FCS-containing 273

media supplemented with increasing extracellular levels of oleate. Importantly, this pattern was also 274

observed in media containing a 1:2:1 palmitate : oleate : linoleate mixture of FAs, representing a 275

physiological mixture that reflects the most prominent circulating free FAs (35). Collectively, these 276

experiments demonstrated that increasing extracellular FA levels enhanced intracellular TAG levels 277

in a range of prostate cancer model systems, and this was not restricted to a specific FA species. 278

Human prostate cancer cells have greater oxidation of extracellular FAs 279

The esterification of extracellular FAs into TAG for storage in lipid droplets is only one 280

intracellular fate for these FAs. Using established radiometric approaches (36), we defined in detail 281

the intracellular handling of extracellular FAs across a range of prostate cancer cells. We observed 282

that FA uptake occurred in all cell lines analyzed, but was faster in LNCaP, C4-2B and PC-3 cells 283




12

(P<0.05), and tended to be slower in 22Rv1 cells (P=0.06) compared to PNT1 cells (Figure 2A). 284

Whilst it has been long assumed that prostate cancer cells activate FA oxidation (10), little direct 285

evidence of catabolism of FAs in prostate cancer cells has been reported. The generation of CO2 286

from extracellular oleate was ~ 11-fold higher in all prostate cancer cells compared to PNT1 cells 287

(Figure 2B). Finally, the incorporation of extracellular FAs into TAG was increased in LNCaP and 288

C4-2B, but lower in 22Rv1 cells compared to PNT1 cells (Figure 2C). 289

Extracellular FAs are the major source of carbons for lipid synthesis in human prostate 290

cancer cells compared to glucose and glutamine 291

The assessment of cancer cell FA metabolism is often limited to the generation of new FAs from 292

non-lipid sources such as glucose and glutamine (i.e. de novo lipogenesis), which has been 293

proposed to meet the increased requirement for phospholipid (37). As such, we next assessed the 294

contribution of glucose and glutamine carbons to total lipid synthesis in a range of prostate cancer 295

cells and non-malignant prostate epithelial cells and compared these to the contribution of 296

extracellular FAs. Firstly, we observed that all cells tested took up glucose and glutamine at a 297

greater rate compared with oleate (Figure 2D-H). Only a very small proportion of the glucose 298

(~5%) and glutamine (~2%) was partitioned to cellular lipids via de novo lipogenesis, whereas the 299

vast majority of oleate was incorporated into lipids (~95%; Figure 2D-H). We further examined the 300

relative contributions of glucose, glutamine and oleate as substrates for total lipid synthesis by 301

summing the absolute rates of lipid synthesis for each. Oleate contributed an average of 83% of 302

carbons to the total lipid pool in all cell lines with glucose providing ~13% and glutamine 303

contributing ~4% (Figure 2I). However, LNCaP cells tended to have a greater contribution to lipid 304

carbons from glucose compared to PNT1 cells (PNT1: 11%; LNCaP: 17%, P=0.1), evidence of 305

increased de novo lipogenesis as previously reported (12-14). Collectively, these data clearly 306

demonstrated that lipid synthesis from glucose and glutamine carbons contributed only a minor 307

fraction (~17%) of the total lipid synthesis in the basal state. 308

Increasing FA availability enhances fatty acid flux into and out of lipid droplets in human 309

prostate cancer cells 310

A major destination for extracellular FAs is TAG stored in cytosolic lipid droplets (Figure 1). This 311

TAG is not a terminal destination for FAs and can be mobilized via the actions of neutral lipases 312

(38,39). As such, we next assessed the catabolism of intracellular-derived FAs. Overnight exposure 313

to media containing higher amounts of oleate and 0.2 µCi/ml 14

C-oleate increased TAG levels in 314

both C4-2B and PC-3 cells compared to cells treated with lower amounts of oleate and 0.2 µCi/ml 315

14C-oleate (data not shown, similar to Figure 1). The oxidation of intracellular TAG-derived FAs 316

was increased in cells that were incubated overnight in media containing more oleate (Figure 2J & 317




13

K). Collectively, these experiments demonstrated that a larger intracellular FA store not only 318

promoted FA oxidation to generate ATP, NADH and other metabolites but also increased 319

mobilization of FAs, potentially for provisioning into phospholipid synthesis. 320

Increasing intracellular TAG levels protects PC-3 cells from apoptosis induced by palmitate 321

or serum starvation 322

We have demonstrated that a range of prostate cancer model systems accumulate lipid droplets, 323

which is supported by recent in vivo data (16). Lipid accumulation is associated with increased 324

tumor burden (11,16) and therefore it is possible that these higher intracellular lipid levels provide a 325

survival advantage. Next we assessed the responsiveness of lipid-loaded castration-resistant C4-2B 326

and PC3 cells to apoptotic stimuli by challenging these cells to either high levels of palmitate in 327

FCS-containing media (21,40,41), or with serum-free media. Both C4-2B and PC3 cells are 328

androgen independent metastatic prostate cancer cell lines that model CRPC, which is currently 329

incurable. The addition of 250 µM palmitate to FCS-containing media reduced MTT absorbance 330

within 2 days and this effect was further enhanced by 4 days compared to cells cultured in FCS-331

containing media (Figure 3A). This reduced metabolic activity was consistent with a striking 332

reduction in cell number after 4 days (Figure 3B) and preceded by activation of PARP and ATF4 333

signaling after 1 day of palmitate treatment (Figure 3C). Interestingly, PC-3 cells lipid-loaded with 334

either oleate alone (Figure 3A), or FA mixture (Figure S1A), were partly protected from palmitate-335

induced apoptosis (Figure 3B) and displayed blunted activation of PARP and ATF4 signaling 336

(Figure 3C). Lipid-loaded PC-3 cells were similarly, but less strikingly, protected from serum 337

starvation-induced reduction in viable cells (Figure S1B) and cell number (Figure S1C). 338

C4-2B cells have low sensitivity to palmitate-induced apoptosis 339

PC-3 and C4-2B cells are castrate-resistant prostate cancer cells that accumulate similar amounts of 340

TAG following incubation in FA-rich media (Figure 1), but only C4-2B cells express AR. C4-2B 341

cells incubated in FCS-containing media supplemented with palmitate had only a mild attenuation 342

of MTT absorbance compared to control cells grown in FCS only media (Figure 3D). This response 343

to palmitate supplementation by C4-2B cells was different from PC-3 cells (Figure 3A), and this 344

difference was also observed in response to dose-dependent palmitate supplementation (Figure 345

S2A-D). Specifically, the MTT absorbance for PC-3 cells was nearly zero after 2 and 4 days of 250 346

µM and 500 µM palmitate supplementation (Figure S2A), whereas 250 µM palmitate 347

supplementation attenuated MTT absorbance of C4-2B cells (D2: 120%, D4: 223% of day 0; Figure 348

2SB) and 500 µM palmitate supplementation reduced MTT absorbance (D2: 77%, D4: 63% of day 349

0; Figure S2B). The blunted MTT signal was due to reduced cell number after 4 days exposure to 350

FCS-containing media supplemented with 250 µM palmitate relative to control, but the number of 351




14

cells were still greater than the number of cells at the start of the experiment (Figure 3E). Similar to 352

PC-3 cells, C4-2B cells pre-treated with either oleate or FA mix were protected from palmitate-353

induced reduction in viable cells (Figure 3D & S1D) and cell number (Figure 3E). Collectively, this 354

demonstrated that PC-3 cells were highly sensitive to palmitate-induced apoptosis whereas C4-2B 355

cells were much less sensitive while lipid-loading both cell lines protected from this palmitate 356

insult. 357

Differences in palmitate handling explain the differential response to palmitate-induced 358

apoptosis in C4-2B and PC-3 cells 359

Altered intracellular palmitate metabolism is one potential explanation for the striking differences in 360

the sensitivity to palmitate-induced lipotoxicity between PC-3 and C4-2B cells, both in the basal 361

state and after lipid loading. To test this, PC-3 and C4-2B cells were incubated in 14

C-palmitate, in 362

the basal state or following overnight exposure to oleate, and the fate of radiolabeled FAs 363

determined. Despite being more sensitive to palmitate-induced apoptosis, PC-3 cells had lower total 364

palmitate uptake compare to C4-2B cells and there was no effect of overnight oleate exposure on 365

palmitate uptake (Figure 4A). Interestingly, C4-2B cells had significantly greater rates of palmitate 366

oxidation compared to PC-3 cells (Figure 4B), which was likely due to increased CPT1 protein 367

levels (Figure 4C). CPT1 catalyzes the rate limiting step in FA oxidation (42). Overnight treatment 368

with oleate induced a modest increase in CPT1 levels in both C4-2B and PC-3 cells (Figure 4C) but 369

this did not change the rate of palmitate oxidation (Figure 4B). 370

The incorporation of palmitate into TAG for storage was also greater in C4-2B cells compared to 371

PC-3 cells (Figure 4D). This difference was not explained by differences in the amount of DGAT-1 372

or DGAT-2 (Figure 4E), which catalyze the final reaction in TAG synthesis (43,44). Pre-treatment 373

with oleate increased TAG synthesis in PC-3 cells up to rates equivalent to C4-2B cells, which did 374

not change in response to oleate (Figure 4D). Additionally, ATGL protein levels were greater in 375

C4-2B cells compared to PC-3 cells, and ATGL expression was increased in both cell lines with 376

overnight oleate treatment (Figure 4E), consistent with increased expression in oleate-treated PDEs 377

(Figure 1B & C) and enhanced lipolysis seen in oleate treated cells (Figure 2J & K). Overall, the 378

partition of FAs between mitochondrial oxidation and storage was similar in C4-2B and PC-3 cells, 379

but this intracellular partitioning of FA was shifted towards storage relative to oxidation in PC-3 380

cells following pre-treatment with oleate (Figure 4F). 381

Palmitate is a critical substrate for de novo ceramide synthesis (45) and one hypothesis to explain 382

the enhanced sensitivity to palmitate in PC-3 cells compared to C4-2B cells was enhanced ceramide 383

synthesis (46). However, the rate of palmitate incorporation into ceramide was lower in PC-3 cells 384




15

compared to C4-2B cells (Figure 4G). Interestingly, overnight oleate exposure reduced ceramide 385

synthesis in C4-2B cells but not in PC-3 cells. There were no differences in the rate of palmitate 386

incorporation in the phospholipid pool (Figure 4H). 387

From these observations, the most striking differences in palmitate metabolism between C4-2B and 388

PC-3 cells were higher palmitate oxidation rates in C4-2B cells and the increase in palmitate 389

incorporation into TAG following overnight oleate treatment in PC-3 cells. These differences in 390

intracellular palmitate handling may explain the differential sensitivity to palmitate-induced 391

apoptosis in C4-2B and PC-3 cells (Figure 3). 392

Inhibition of mitochondrial FA oxidation sensitizes C4-2B cells to palmitate-induced apoptosis 393

Another explanation for the observed resistance of C4-2B cells to palmitate-induced apoptosis 394

compared to PC-3 cells may be higher palmitate oxidation and higher CPT1A protein levels (Figure 395

4B & C). Therefore, we tested whether inhibiting CPT1-mediated palmitate oxidation sensitized 396

C4-2B cells to palmitate-induced apoptosis. Treating cells with 100 µM of the CPT1 inhibitor, 397

etomoxir, lowered palmitate oxidation (Figure 5A). The addition of 250 µM palmitate to growth 398

media reduced cell viability (Figure 5B); however, the combination of etomoxir and palmitate 399

completely abolished MTT metabolic activity (Figure 5B), cell number (Figure 5C), and activated 400

PARP signaling (Figure 5D). Similar, but less striking, patterns were observed in siRNA-mediated 401

CPT1A knockdown in C4-2B cells. Knockdown of CPT1A (Figure 5E) lowered palmitate oxidation 402

(Figure 5F) but this was associated with a mild reduction in MTT metabolic activity (Figure 5G) 403

and no effect on cell number (Figure 5H). The addition of 250 µM palmitate to growth media 404

attenuated cell viability (Figure 5G); however, the combination of CPT1A knockdown and 405

palmitate further lowered MTT metabolic activity (Figure 5G) and cell number (Figure 5H). 406

Collectively, these results indicate that inhibition of FA oxidation sensitized C4-2B cells to 407

palmitate-induced apoptosis. 408

Inhibition of oleate-stimulated TAG synthesis restores sensitivity to palmitate-induced 409

apoptosis in lipid-loaded PC-3 cells 410

Pre-treatment with either oleate alone or the FA mixture protected PC-3 cells from palmitate-411

induced and serum starvation-induced apoptosis (Figure 3 & S1). Radiometric analysis of palmitate 412

metabolism pointed to an increase in TAG synthesis to shunt palmitate into lipid droplets (Figure 413

4D) as a potential mechanism by which pre-treatment with FAs protected PC-3 cells from 414

palmitate-induced apoptosis. We directly tested this by inhibiting TAG synthesis through the 415

addition of a DGAT-1 inhibitor only during the oleate pre-incubation of PC-3 cells prior to 416

palmitate treatment. As expected, DGAT-1 inhibition blunted oleate-induced increase in PC-3 TAG 417




16

content (Figure 6A) due to reduced incorporation of radiolabeled oleate into TAG (Figure 6B). As 418

previously observed, palmitate treatment induced apoptosis, as determined by reduced MTT (Figure 419

6C), PARP activation (Figure 6D) and reduced cellular protein content (Figure 6E). Pre-incubation 420

with oleate blunted this effect (Figure 6C-E); however, lowering intracellular TAG levels by DGAT 421

inhibition in the presence of oleate restored PC-3 cell sensitivity to palmitate (Figure 6C-E). 422

Importantly, pre-incubation with DGAT-1 inhibitor did not affect subsequent PC-3 (Figure S3) and 423

C4-2B (data not shown) cell growth in FCS-containing media. Therefore, TAG synthesis is required 424

for the protective effects of pre-treating PC-3 cells with FAs to palmitate-induced apoptosis. 425




17

DISCUSSION 426

Cancer cells require adaptive alterations in intermediary metabolism to fulfil the energy 427

requirements and biochemical needs of their uncontrolled growth capacity (47). Unlike many other 428

solid tumors, prostate cancer exhibits a low rate of glucose utilization and an increased dependence 429

on lipids as a major energy source (48). While enhanced de novo fatty acid synthesis in prostate 430

cancer has been firmly established (see review (23)), the crucial importance of extracellular FAs in 431

prostate cancer progression has been underappreciated and less well-studied. Using a range of 432

models, we demonstrate that prostate cancer cells take up FAs and incorporate them into 433

intracellular TAG for storage in a dose-dependent manner. Further, these extracellular FAs are a 434

greater contributor to intracellular synthesis compared to glucose and glutamine which are used as 435

substrates for de novo fatty acid synthesis. We also show for the first time, striking heterogeneity in 436

the intracellular handling of FAs in castration-resistant C4-2B and PC-3 prostate cancer cells and in 437

their response to palmitate-induced apoptosis. Specifically, C4-2B cells have increased FA 438

oxidative capacity that underpins their resistance to palmitate-induced apoptosis. On the other hand, 439

PC-3 cells are highly sensitive to palmitate-induced apoptosis, which was inhibited by prior lipid-440

loading to stimulate TAG synthesis. These observations highlight the diversity of intracellular FA 441

metabolism in prostate cancer cells. 442

Storage of lipids is an evolutionarily-conserved phenomenon which can buffer energy fluctuations 443

and promote survival in all cells and organisms (49). In this scenario, lipid storage predominantly 444

refers to the partitioning of excess FAs into TAG for storage in cytosolic lipid droplets. Lipid 445

droplets are closely localized with most intracellular organelles, notably mitochondria and 446

endoplasmic reticulum (ER) and are highly conserved in yeast through to mammals (50) but the 447

size and number of lipid droplets varies between cell types. Prostate cancer cells have detectable 448

levels of TAGs and lipid droplets (11,51) and the amounts of these correlate with disease grade 449

(11). Here, we show that PDEs, LNCaP and C4-2B spheroids and a range of prostate cancer cells 450

can respond to changing levels of extracellular FAs and accumulate these as TAG in a dose-451

dependent manner, independent of FA species. Collectively, these in vitro observations suggest that 452

the lipid biology of prostate cancer is likely to be influenced by the local lipid environment of the 453

host, including local adipose tissue that may be expanded in obese patients and/or the circulating 454

lipid profile which itself is influenced by diet and body composition (52,53). 455

Several studies have reported that exogenous FA availability can influence prostate cancer cell 456

growth in vitro (54-60). Monounsaturated FAs, including oleate (C18:1), stimulate LNCaP cell 457

proliferation (54) but activate apoptosis in DU145 cells (60) and retard growth in PC-3 cells 458

(58,59). However, another study reported that oleate stimulates PC-3 but not LNCaP cell growth 459




18

(55), which cannot be explained by differences in concentration. Similar inconsistent observations 460

have been made using other FA species including linoleate (C18:2), arachidonic acid (C20:4), and 461

eicosapentaenoic acid (C20:5) (54,55,59,60). Despite these inconsistencies, overall these 462

observations suggest that FAs can have both pro- or anti-proliferative effects. Surprisingly, little is 463

known about the effects of extracellular palmitate (C16:0), which accounts for ~20-22% of FA 464

species of complex lipids (i.e. phospholipids, TAGs etc.) (61) or ~30% of free FAs (62) in human 465

plasma, on prostate cancer cells. The saturated FA palmitate can induce apoptosis in a range of cells 466

including 3T3 fibroblasts (41), peripheral blood mononuclear cells (63), human cardiac progenitor 467

cells (64), pancreatic β cells (65,66), macrophages (67), breast cancer cells (21,40) and hepatocytes 468

(68). We observed that palmitate indeed activates apoptosis in PC-3 cells but attenuates C4-2B cell 469

growth. This is similar to our recent observations in breast cancer cells, where palmitate induced 470

apoptosis in triple negative MDA-MB-231 cells but only attenuated growth in estrogen receptor α 471

positive MCF-7 cells (21). The precise mechanism by which palmitate induces apoptosis remains to 472

be elucidated but several mechanisms have been proposed. These include ER stress (65), impaired 473

autophagy (63), altered NAD metabolism (68), and ceramide synthesis (66). 474

Prostate cancer is initially sensitive to hormonal manipulation; however, resistance to androgen 475

deprivation therapy ultimately occurs which results in the development of lethal metastatic 476

castration-resistant prostate cancer. Here, we identified that the differential responsiveness of 477

castration-resistant C4-2B and PC-3 cells to palmitate was associated with differences in palmitate 478

handling. Specifically, attenuation of C4-2B cell growth by palmitate treatment, rather than 479

activation of apoptosis, was due to enhanced FA oxidation compared to PC-3 cells. FA oxidation 480

has been proposed to be a dominant bioenergetic pathway in prostate cancer cells (10). Whilst the 481

relative contribution of various substrates to ATP turnover in prostate cancer cells is yet to be 482

reported, we provide clear evidence that prostate cancer cells have increased FA oxidation 483

compared to prostate epithelial PNT-1 cells. Our data compliment a previous observation that 484

LNCaP and VCaP prostate cancer cells have greater palmitate oxidation compared to the benign 485

prostatic hyperplasia epithelial cell line BPH-1 (69). We also show that the differences between C4-486

2B and PC-3 cells may be related to CPT1A expression and function. Inhibition of FA oxidation by 487

etomoxir and CPT1A knockdown in C4-2B cells attenuated growth, consistent with a previous 488

observation in castration-sensitive LNCaP and VCaP cells and xenografts (69). Interestingly, the 489

attenuation of C4-2B cell growth by the CPT1A inhibitor etomoxir was similar to palmitate 490

treatment, with the combination of FA oxidation inhibition and palmitate treatment leading to cell 491

death. Our results shed new light on the contribution of FA oxidation in castration-resistant C4-2B 492

cells where pharmacological and genetic inhibition of FA oxidation sensitized C4-2B cells to 493




19

palmitate-induced apoptosis and in combination with other observations (69) suggests that targeting 494

FA oxidation is an attractive therapeutic strategy in prostate cancer. 495

The saturated FA palmitate can activate apoptosis in a range of cells (21,40,41,63-68,70), via a 496

range of proposed mechanism, and we show that this also occurs in PC-3 prostate cancer cells. 497

Another common observation is that the addition or presence of oleate ameliorates the cytotoxic 498

effects of palmitate (67,68,71-74). Interestingly, oleate also prevents arachidonic acid and linoleic 499

acid induced cell death in DU-145 prostate cancer cells (60). Several mechanisms have been 500

proposed including restoring insulin stimulated protein kinase B (Akt) signaling (73), attenuating 501

palmitate-induced ER stress (71), preventing activation of the unfolded protein response (75), 502

activating pro-survival pathways of ER stress (72), and activation of AMP-activated protein kinase 503

and mechanistic target of rapamycin signaling (74). Co-treatment with oleate can also modulate 504

palmitate metabolism to increase TAG synthesis and prevent diacylglycerol accumulation (73,76), 505

and block mitochondrial dysfunction and production of reactive oxygen species (76). We observed 506

that pre-treatment of PC-3 cells with either oleate or a FA mixture prevented palmitate-induced 507

apoptosis, as well as serum starvation, by increasing palmitate partitioning into TAG synthesis for 508

storage in lipid droplets. We also observed that pre-treatment of C4-2B cells with either oleate or a 509

FA mixture prevented palmitate-induced attenuation of growth. The ability of FA pre-treatment to 510

alter the response to palmitate treatment by PC-3 and C4-2B cells was not due to changes in 511

palmitate uptake. We did observe a reduction in palmitate incorporation into ceramide in C4-2B 512

cells; however, C4-2B cells have higher rates of ceramide synthesis from palmitate compared to 513

PC-3 cells despite C4-2B cells being less sensitive to the cytotoxic effects of palmitate. This 514

suggests that ceramide synthesis does not activate apoptosis in these cells and that the preventative 515

ability of FA pre-treatment was due to altered TAG synthesis and oxidation. 516

We and others have demonstrated that TAG synthesis can protect cells from palmitate-induced 517

lipotoxicity (21,41,77). For example, oleate supplementation promotes TAG synthesis in CHO cells 518

that can prevent palmitate-induced apoptosis in mouse embryonic fibroblasts but this protection was 519

not seen in DGAT1-/-

mouse embryonic fibroblasts, which lack the ability to synthesize TAG (78). 520

Recently, we demonstrated that TAG synthesis was required for oleate to protect MDA-MB-231 521

breast cancer cells from palmitate-induced apoptosis (21) and here we show that this also occurs in 522

PC-3 cells. The final step of TAG synthesis is catalyzed by DGAT1 and DGAT2 and knockdown of 523

DGAT1 in LNCaP cells reduced cell growth and colony formation (79). Collectively, these 524

observations demonstrate that TAG synthesis supports cancer cell progression. Interestingly, the 525

inability to breakdown TAG to liberate FAs also impairs cell growth and invasion in LNCaP cells 526

(51,79). TAG hydrolysis is catalyzed by ATGL and knockdown of ATGL in LNCaP cells ablated 527




20

cell invasion and growth (51). We observed that pre-treatment with oleate increased ATGL protein 528

levels as well as oxidation of TAG-derived FAs in both C4-2B and PC-3 cells. Collectively, these 529

observations suggest that TAG-derived FAs may support prostate cancer cell progression and 530

therefore suggest that FAs that traverse lipid droplet contained TAG pool play an important role in 531

prostate cancer biology. 532

In conclusion, we report for the first time that prostate cancer cells can use extracellular FAs as both 533

fuel for oxidation and the primary substrates for complex lipid synthesis such as TAG, and that high 534

extracellular lipid availability further enhances FA flux in these cells. Further, we identified distinct 535

differences in palmitate handling and sensitivity between castration-resistant C4-2B and PC-3 cells. 536

The reduced sensitivity of C4-2B cells to palmitate-induced apoptosis was due to the high rates of 537

FA oxidation, thus suggesting a potential therapeutic vulnerability for AR positive prostate cancer 538

types with high levels of CPT1A. Oleate pre-treatment, which stimulated TAG synthesis, prevented 539

palmitate-induced apoptosis in AR negative PC-3 cells and adds to a growing body of evidence 540

suggesting that proteins regulating intracellular TAG homeostasis may be further therapeutic 541

targets. The outcomes from these experiments inform the potential role that obesity-associated 542

dyslipidemia (see review (23)) or the host circulating lipidome (24) may play in influencing prostate 543

cancer progression and therefore, these metabolic traits might be the basis for novel, targeted 544

treatment interventions in prostate cancer. 545




21

LIST OF ABBREVIATIONS 546

AR: androgen receptor; ATF4: activating transcription factor 4; ATGL: adipose triglyceride lipase; 547

BSA: bovine serum albumin; CPT1A: carnitine palmitoyltransferase 1A; DGAT-1: diacylglycerol 548

O-acyltransferase 1; DGAT-2: diacylglycerol O-acyltransferase 2; ER: endoplasmic reticulum; FA: 549

fatty acid; FCS: fetal calf serum; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; PARP: poly 550

(ADP ribose) polymerase; PDE: patient-derived explant; TAG: triacylglycerol; TLC: thin layer 551

chromatography 552

Authors’ contributions 553

SB, ZDN, AYZ, EHB, MMC, MS, HML, AA, BV, FLF, LSL, RFS, RAH, NLR and MSK 554

performed experiments and analyzed data. DNS, JH, LGH and LMB provided intellectual input, 555

designed experiments, analyzed data and edited the manuscript. AJH conceived the general ideas 556

for this study, designed and performed experiments, analyzed data and wrote the manuscript. All 557

authors read and approved the final version of the manuscript. 558

Acknowledgments 559

Thanks to the Bosch Institute Molecular Biology Facility for technical support. 560




22

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76. Kwon B, Lee HK, Querfurth HW. Oleate prevents palmitate-induced mitochondrial 781

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795




30

FIGURES 796

Figure 1: Prostate cancer cells, spheroids and patient-derived tumor explants take up and 797

store FA as TAG. Patient-derived tumor explant (A) neutral lipid levels by oil red-O staining, (B) 798

representative image and (C) intensity of ATGL immunostaining following 72 h incubation in 799

media containing 500 µM oleate, where 0 = no staining, 1 = 1+ (moderate) immunostaining, and 2 800

= 2+ (high) immunostaining, and (D) 3H-oleate uptake. Scale bars = 100 µm (A) and 200 µm (B). 801

Oil red-O staining is representative of n=5 patient-derived explants, and ATGL IHC is 802

representative of n=5 patient-derived explants that contained cancer. ATGL IHC quantification is 803

paired for multiple explants, P determined by Paired Student’s t-test. 3H-oleate uptake data is paired 804

for multiple explants from 3 individuals (Patient 1 (empty circles), Patient 2 (grey circles), and 805

Patient 3 (filled circles). * P ≤ 0.05 main effect for time; # P ≤ 0.05 main effect for insulin by two-806

way ANOVA. LNCaP spheroid (E) neutral lipid levels by oil red-O staining and (F) TAG content 807

following 72 h incubation in oleate. C4-2B spheroid (G) neutral lipid levels by oil red-O staining 808

and (H) Triacylglycerol (TAG) content following 72 h incubation in 0.5 mM oleate. Scale bars = 809

200 µm. Oil red-O staining is representative of n=18 spheroids. TAG data is presented as single 810

measure of n=18 spheroids combined. TAG content of (I) PNT1, (J) LNCaP, (K) C4-2B, (L) 811

22Rv1, and (M) PC-3 cells following overnight incubation in either oleate alone or 1:2:1 mixture of 812

palmitate:oleate:linoleate (FA Mix; three (PNT1, 22Rv1, LNCaP) or five (C4-2B, PC-3) 813

independent experiments performed in triplicate). Data are presented as mean ± SEM. * P ≤ 0.05 814

for main effect for [FA]; # P ≤ 0.05 vs. Oleate at same [FA] by two-way ANOVA followed by 815

Tukey’s Multiple Comparisons test. 816

Figure 2 Comparison of substrate metabolism in a range of prostate cancer cells. 14

C-oleate 817

(A) uptake, (B) oxidation, and (C) incorporation into TAG in PNT1, LNCaP, C4-2B, 22Rv1, and 818

PC-3 cells (three independent experiments performed in duplicate). (D-H) Absolute rates of 14

C-819

labeled substrate incorporation into intracellular lipids (lipid synthesis) and total uptake (sum of 820

media 14

CO2, 14

C activity in both the aqueous and organic phases of a Folch extraction) of various 821

substrates in (D) PNT-1, (E) LNCaP, (F) C4-2B, (G) 22Rv1, and (H) PC-3 cells and (I) percent 822

contribution of substrates to lipid synthesis in all cell lines in the basal state (three independent 823

experiments performed in duplicate). Total lipid synthesis was defined as the sum of the grey bars 824

in panels D-H. Oxidation of TAG-derived 14

C-oleate in (J) C4-2B and (K) PC-3 cells following 825

overnight incubation in either low (20 or 80 µM oleate respectively) or high (150 or 300 µM oleate 826

respectively) (three independent experiments performed in triplicate). Data are presented as mean ± 827

SEM. A-C * P ≤ 0.05 vs. PNT1 by One-way ANOVA followed by Dunnett's multiple comparisons 828




31

test, D-H * P ≤ 0.05 vs. Oleate by Two-way ANOVA followed by Tukey’s Multiple Comparisons 829

test. J, K * P ≤ 0.05 vs. 20 or 80 µM respectively by Student’s t-test. 830

Figure 3 PC-3 cells are sensitive to palmitate-induced apoptosis but C4-2B cells are not, and 831

lipid-loading protects PC-3 cells from palmitate-induced apoptosis. (A) MTT assays and (B) 832

cell number of PC-3 cells incubated in FCS-containing media supplemented with 250 μM palmitate 833

for 4 days with or without prior overnight incubation with oleate or 1:2:1 mixture of 834

palmitate:oleate:linoleate (FA Mix). MTT results are presented as percentages of MTT absorbance 835

at indicated time points relative to that at day 0 for each group. The dashed line represents the 836

number of cells present at day 0 (MTT: five independent experiments performed in quadruplicate; 837

Cell count: three independent experiments performed in duplicate). (C) Representative 838

immunoblots of cPARP and ATF4 levels of PC-3 cells incubated in FCS-containing media 839

supplemented with 250 μM palmitate for 1 day with or without prior overnight incubation with 840

oleate (representative of three independent experiments performed in triplicate). (D) MTT assays 841

and (E) cell number of C4-2B cells incubated in FCS-containing media supplemented with 250 μM 842

palmitate for 4 days with or without prior overnight incubation with oleate. MTT results are 843

presented as percentages of MTT absorbance at indicated time points relative to that at day 0 for 844

each group. The dashed line represents the number of cells present at day 0 (MTT: three 845

independent experiments performed in quadruplicate; Cell count: three independent experiments 846

performed in duplicate). Data are presented as mean ± SEM. * P ≤ 0.05 vs. Palmitate; # P ≤ 0.05 vs. 847

Control by Two-way ANOVA (A & D) or One-way ANOVA (B & E) followed by Tukey’s 848

Multiple Comparisons test. 849

Figure 4: PC-3 and C4-2B cells metabolize palmitate differently and this is selectively altered 850

by pre-treatment with oleate. 14

C-palmitate (A) uptake and (B) oxidation in C4-2B and PC-3 cells 851

with or without prior overnight incubation with 150 µM oleate. (C) Representative immunoblots 852

and densitometric quantitation of CPT1A in C4-2B and PC-3 cells with or without prior overnight 853

incubation with oleate. (D) 14

C-palmitate incorporation into triacylglycerol (TAG) in C4-2B and 854

PC-3 cells with or without prior overnight incubation with oleate. (E) Representative immunoblots 855

of DGAT1, DGAT2 and ATGL, and densitometric quantitation of ATGL in C4-2B and PC-3 cells 856

with or without prior overnight incubation with oleate. (F) Intracellular partitioning of 14

C-palmitate 857

as expressed as the ratio of 14

C-palmitate incorporation into triacylglycerol (storage) vs. 14

C-858

palmitate oxidation in C4-2B and PC-3 cells with or without prior overnight incubation with oleate. 859

14C-palmitate incorporation into (G) ceramide and (H) phospholipid in C4-2B and PC-3 cells with 860

or without prior overnight incubation with oleate. Data are presented as mean ± SEM of three 861

independent experiments performed in triplicate. † P ≤ 0.05 main effect for cells; * P ≤ 0.05 vs. - 862




32

Oleate; # P ≤ 0.05 vs. C4-2B cells - Oleate by Two-way ANOVA followed by Tukey’s Multiple 863

Comparisons test. 864

Figure 5: Inhibition of fatty acid oxidation in C4-2B cells results in sensitization to palmitate-865

induced apoptosis. (A) 14

C-palmitate oxidation in C4-2B cells that were treated with or without 866

100 µM Etomoxir (Eto) (three independent experiments performed in triplicate). (B) MTT assays 867

and (C) cell number of C4-2B cells incubated in FCS-containing media supplemented with 250 μM 868

palmitate (Palm), Etomoxir (Eto) or a combination for 4 days. MTT results are presented as 869

percentages of MTT absorbance at indicated time points relative to that at day 0 for each group. The 870

dashed line represents the number of cells present at day 0 (MTT: four independent experiments 871

performed in quadruplicate; Cell count: three independent experiments performed in duplicate). (D) 872

Representative immunoblots of cPARP levels of C4-2B cells incubated in FCS-containing media 873

supplemented with 250 μM palmitate, Etomoxir or a combination for 1 days (representative of three 874

independent experiments performed in triplicate). (E) Representative immunoblots of CPT1A of 875

C4-2B cells treated with Control or CPT1A siRNA for 2 days (representative of three independent 876

experiments performed in triplicate). (F) 14

C-palmitate oxidation in C4-2B cells treated with or 877

without CPT1A siRNA for 4 days (three independent experiments performed in triplicate). (G) 878

MTT assays and (H) cell number of C4-2B cells treated with Control or CPT1A siRNA for 2 days 879

then incubated in FCS-containing media with or without supplementation with 250 μM palmitate 880

for 4 days. MTT results are presented as percentages of MTT absorbance at indicated time points 881

relative to that at day 0 for each group. The dashed line represents the number of cells present at day 882

0 (MTT: four independent experiments performed in quadruplicate; Cell count: three independent 883

experiments performed in duplicate). Data are presented as mean ± SEM. (A) * P ≤ 0.05 vs. Control 884

by Unpaired Student’s t-test. (B, C) * P ≤ 0.05 vs. palmitate; # P ≤ 0.05 vs. etomoxir by two-way 885

ANOVA followed by Tukey’s Multiple Comparisons test. (F) * P ≤ 0.05 vs. Control by Unpaired 886

Student’s t-test. (G, H) * P ≤ 0.05 vs. palmitate; # P ≤ 0.05 vs. CPT1A KD by two-way ANOVA 887

followed by Tukey’s Multiple Comparisons test. 888

Figure 6: Inhibition of TAG synthesis in PC-3 cells blunts the protective effects of prior oleate 889

treatment to palmitate-induced apoptosis. (A) PC-3 cell triacylglycerol (TAG) levels in cells 890

treated with 150 µM oleate (Ol) with or without 60 nM DGAT inhibitor AZD3988 (iDGAT) for 24 891

h (three independent experiments performed in duplicate). (B) 14

C-oleate incorporation into TAG in 892

PC-3 cells that were treated with or without DGAT inhibitor (three independent experiments 893

performed in duplicate). (C) MTT assays of PC-3 cells incubated in FCS-containing media 894

supplemented with 250 μM palmitate (Palm) for 4 days following prior incubation with 150 µM 895

oleate (Ol) with or without 60 nM DGAT inhibitor AZD3988 (iDGAT). MTT results are presented 896




33

as percentages of MTT absorbance at indicated time points relative to that at day 0 for each group 897

(three independent experiments performed in quadruplicate). (D) Representative immunoblots of 898

cPARP of PC-3 cells incubated in FCS-containing media supplemented with 250 μM palmitate for 899

1 day following prior incubation with oleate with or without DGAT inhibitor (three independent 900

experiments performed in triplicate). (E) Protein levels in PC-3 cells incubated in FCS-containing 901

media supplemented with 250 μM palmitate for 4 days following prior incubation with 150 µM 902

oleate with or without DGAT inhibitor (three independent experiments performed in triplicate). 903

Data are presented as mean ± SEM. (A) * P ≤ 0.05 vs. Control; # P ≤ 0.05 vs. Oleate by One-way 904

ANOVA followed by Tukey’s Multiple Comparisons test. (B) # P ≤ 0.05 vs. Oleate by Student’s t-905

test. (C) * P ≤ 0.05 vs. Palmitate; # P ≤ 0.05 vs. Control, $ P ≤ 0.05 vs. Palm + Oleate by Two-way 906

ANOVA followed by Tukey’s Multiple Comparisons test. (E) * P ≤ 0.05 vs. Palmitate; # P ≤ 0.05 907

vs. Control, $ P ≤ 0.05 vs. Palm + Oleate by One-way ANOVA followed by Tukey’s Multiple 908

Comparisons test. 909




PNT1

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Figure 2




A B C

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Palmitate

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Figure 3




A B C D

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Figure 4




A B C D

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Figure 5




A B CCC

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Figure 6




Published OnlineFirst January 15, 2019.Mol Cancer Res Seher Balaban, Zeyad D Nassar, Alison Y. Zhang, et al. Synthesis in Prostate CancerExtracellular Fatty Acids are the Major Contributor to Lipid

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