Inhibition of fatty acid desaturation is detrimental to cancer cell survival in metabolically compromised environments
- Barrie Peck†1, 12Email author,
- Zachary T. Schug†2,
- Qifeng Zhang3,
- Beatrice Dankworth4,
- Dylan T. Jones5,
- Elizabeth Smethurst3,
- Rachana Patel2,
- Susan Mason2,
- Ming Jiang6,
- Rebecca Saunders6,
- Michael Howell6,
- Richard Mitter7,
- Bradley Spencer-Dene8,
- Gordon Stamp8,
- Lynn McGarry2,
- Daniel James2,
- Emma Shanks2,
- Eric O. Aboagye9,
- Susan E. Critchlow10,
- Hing Y. Leung2,
- Adrian L. Harris5,
- Michael J. O. Wakelam3,
- Eyal Gottlieb2 and
- Almut Schulze1, 4, 11Email author
© Peck et al. 2016
Received: 22 July 2015
Accepted: 7 March 2016
Published: 1 April 2016
Enhanced macromolecule biosynthesis is integral to growth and proliferation of cancer cells. Lipid biosynthesis has been predicted to be an essential process in cancer cells. However, it is unclear which enzymes within this pathway offer the best selectivity for cancer cells and could be suitable therapeutic targets.
Using functional genomics, we identified stearoyl-CoA desaturase (SCD), an enzyme that controls synthesis of unsaturated fatty acids, as essential in breast and prostate cancer cells. SCD inhibition altered cellular lipid composition and impeded cell viability in the absence of exogenous lipids. SCD inhibition also altered cardiolipin composition, leading to the release of cytochrome C and induction of apoptosis. Furthermore, SCD was required for the generation of poly-unsaturated lipids in cancer cells grown in spheroid cultures, which resemble those found in tumour tissue. We also found that SCD mRNA and protein expression is elevated in human breast cancers and predicts poor survival in high-grade tumours. Finally, silencing of SCD in prostate orthografts efficiently blocked tumour growth and significantly increased animal survival.
Our data implicate lipid desaturation as an essential process for cancer cell survival and suggest that targeting SCD could efficiently limit tumour expansion, especially under the metabolically compromised conditions of the tumour microenvironment.
KeywordsLipid desaturation Breast cancer Prostate cancer Lipidomics SCD
Altered metabolism has now been established as a central hallmark of oncogenic transformation . Aberrant activation of oncogenic signalling pathways and loss of tumour suppressor function alters metabolic processes in cancer cells to satisfy their increased energetic and biosynthetic demand [2, 3]. Moreover, the reduced availability of nutrients and oxygen in poorly vascularised regions of solid tumours also affects the metabolic activity of cancer cells [2, 4]. This metabolic reprogramming supports cancer proliferation and survival but renders cancer cells highly susceptible to perturbations within the metabolic network .
Lipid metabolism is frequently altered in human cancer [6, 7]. While most normal adult tissues use dietary lipids provided by the blood stream, many cancers show increased rates of de novo fatty acid (FA) biosynthesis . The expression of most enzymes involved in FA biosynthesis is controlled by the sterol regulatory element binding proteins (SREBPs) and can be induced downstream of the PI3-kinase/Akt/mTORC1 signalling pathway in cancer . Consequently, de novo FA biosynthesis is generally high in those tumour types that exhibit activating mutations within this pathway, including breast and prostate cancers [10, 11].
Several enzymes within the FA biosynthesis pathway have been found to be essential for cancer cell growth and survival and are currently pursued as targets for therapeutic development . The potential selectivity of targeting lipid metabolism in cancer was also supported by a systems biology study employing a genome scale model of cancer metabolism that predicted specific dependence of cancer cells on lipid metabolism enzymes . This suggests that targeting enzymes involved in these processes should selectively interfere with the growth of cancer cells without overt toxicity towards normal tissues.
Here, we show that cancer cells are highly dependent on stearoyl-CoA desaturase (SCD), the enzyme that introduces a double bond at the Δ9 position of newly synthesised FAs. Loss of cell number was only detected under lipid-depleted conditions and fully restored by exogenous mono-unsaturated FAs. Depletion of SCD caused specific alterations in lipid composition, indicating reduced availability of unsaturated acyl chains for the synthesis of phosphoglycerides and cardiolipins. Inhibition of SCD led to the release of cytochrome C from the mitochondria leading to the induction of apoptosis and increased sensitivity towards cytotoxic drugs and inhibitors of mitochondrial respiratory complexes. The sensitivity of cancer cells towards SCD inhibition was greatly increased by spheroid culture, a condition that recapitulates nutrient and oxygen gradients found in tumours. Expression of SCD is enhanced in human breast cancer tissue and associated with high disease grade. Finally, silencing of SCD resulted in efficient reduction of tumour growth in prostate orthografts. Together, these results indicate that SCD is an important node in the metabolism of cancer cells and that inhibition of this enzyme could provide a successful strategy for cancer treatment.
Antibodies and reagents
SCD inhibitors were purchased from Biovision and Caymen chemicals. Anti-SCD antibodies (SCD11-A) were from Alpha Diagnostics International and anti-Akt (9272), anti-cytochrome C (11940S) and anti-PARP (9542) from Cell Signalling. Hydrocortisone, EGF, Akt inhibitor (Akt V) and staurosporin were from Calbiochem. Insulin, cholera toxin, puromycin, doxycycline, paclitaxel and rotenone were from Sigma-Aldrich and metformin from Tocris Biosciences.
RWPE1 and LNCaP (clone FGC) were obtained from the American Type Culture Collection. All other cell lines were obtained from LRI Cell Services (CRUK LRI, London, UK). All cell lines were authenticated using STR profiling and used at low passage. RWPE1 cells were grown in keratinocyte serum-free medium (Gibco) supplemented with epidermal growth factor and bovine pituitary extract (KGM). DU145, LNCaP and PC3 cells were grown in RPMI supplemented with 2 mM L-glutamine and penicillin/streptomycin. All breast cancer cell lines were grown in DMEM/F12 supplemented with serum, glutamine and penicillin/streptomycin. MCF10a cells were grown in DMEM/F12 supplemented with 5 % horse serum, 20 ng/ml EGF, 5 μg/ml hydrocortisone, 10 μg/ml insulin and 100 ng/ml cholera toxin.
Breast and prostate cancer cells were reverse-transfected with 37.5 nM of Dharmacon siGENOME siRNA using Lullaby reagent (Oz biosciences). After 24 h, culture medium was replaced with either 10 % (full serum) or 1 % (low serum) foetal calf serum (FCS) containing medium. Additional supplementations were included for the experiments indicated. After 72 h, cells were fixed in 80 % ethanol over night at −20 °C. Plates were subsequently stained with DAPI (Sigma), and cell number was determined using the ACUMEN X3 microplate cytometer.
Generation of doxycycline-inducible TetOnPLKO-shRNA cell lines
shRNA sequences targeting SCD or non-targeting control (NTC) were cloned into the TetOn-pLKO-puro lentiviral vector . Clone IDs for shRNAs are as follows: shSCD #1 (TRCN0000056613) and shSCD #2 (TRCN0000056614). Lentiviruses were produced by cotransfecting HEK293T cells with lentiviral and packaging plasmids pCMVΔR8.91 and pMD.G. Supernatants were collected 72 h after transfection, mixed with polybrene (8 μg/mL) and used to infect cells. Cells were selected in medium containing puromycin (2 μg/mL).
RNA extraction, reverse transcription, RT-qPCR
Total cell RNA was extracted using an RNeasy kit (QIAGEN); 2 μg of RNA was utilized for first strand cDNA synthesis with oligo-dT primers and Superscript II Reverse Transcriptase (Invitrogen). RT-qPCR was performed using SYBR® Green PCR Master Mix (Applied Biosystems) and Quantitect primers (QIAGEN) on an ABI Prism 7900 (Applied Biosystems). All reactions were performed in duplicate, and relative mRNA expression was calculated using the comparative Ct method after normalization to the loading control B2M.
Cells were lysed in Triton lysis buffer (1 % Triton X100, 50 mM Tris pH7.5, 300 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM NaVO4, Protease-Inhibitor-Cocktail and Phosphatase-Inhibitor-Cocktail (Roche)). Proteins were separated on SDS-PAGE and blotted onto PVDF membrane (Immobilon). Membranes were blocked with 3 % bovine serum albumin (BSA) and incubated with antibody solutions, and signals were detected using ECL-reagent.
Stable isotope labelling was performed as in . For lipidomic analysis, lipids were extracted using a methanol/chloroform extraction method and quantified by LC-MS analysis on a Shimadzu IT-TOF LC/MS/MS system. Accurate mass (with mass accuracy ~5 ppm) and tandem MS were used for molecular species identification and quantification. The identity of lipids was further confirmed by reference to appropriate lipid standards. Cell pellets were spiked with appropriate internal standards (for each sample, 100 ng 12:0/12:0/12:0-TG, 200 ng 12:0/12:0-DG, 100 ng 12:0-MG, 200 ng 17:0-FA, 100 ng C17-Cer, 50 ng C17-SG, 200 ng 14:0/14:0/14:0/14:0-CL, 100 ng 12:0/12:0-PG, 200 ng 12:0/12:0-PE, 200 ng 12:0/12:0-PS, 400 ng 17:0/20:4-PI, 100 ng 12:0/12:0-PA, 400 ng 12:0/12:0-PC, 100 ng 17:0-LPA, 100 ng 17:0-LPC, 100 ng 12:0-Cer1P, 100 ng C17-S1P, 200 ng C17-SM and 50 ng C17-SPC) before extraction. The samples were extracted using a modified Folch method: first extraction with 4 ml chloroform:2 ml methanol:2 ml 0.88 % NaCl for each sample and second extraction of upper phase with 3 ml of synthetic lower phase of chloroform/methanol/0.88 % NaCl 2:1:1; the combined lower phases of the lipid extract were dried using a Thermo SpeedVac at room temperature under vacuum and re-dissolved in 50 μl chloroform/methanol 1:1, of which 7 μl was injected onto the column for LC-MS analysis. For LC/MS/MS analysis, a Shimadzu IT-TOF LC/MS/MS system hyphenated with a five-channel online degasser, four-pump, column oven, and autosampler with cooler Prominence HPLC (Shimadzu) was used. In detail, lipid classes were separated on a normal phase silica gel column (2.1 × 150mm, 4micro, MicoSolv Technology) using a hexane/dichloromethane/chloroform/methanol/acetanitrile/water/ethylamine solvent gradient based on the polarity of head group. Accurate mass (with mass accuracy ~5 ppm) and tandem MS were used for molecular species identification and quantification. The identity of lipid was further confirmed by reference to appropriate lipid standards. IT-TOF mass spectrometer operation conditions: ESI interface voltage +4.5 kv for positive ESI and −4 kv for negative ESI, heat block temperature 230 °C, nebulising gas flow 1.4 L/min, and CDL temperature 210 °C, with drying gas on at pressure of 100 kPa. All solvents used for lipid extraction and LC/MS/MS analysis were LC-MS grade from Fisher Scientific. Lipid amounts were normalised by protein concentrations of each sample.
Crystal violet staining
Cells were seeded on 12-well plates. After incubation, cells were fixed with 70 % ethanol, stained with 0.01 % crystal violet. For quantification, dye was extracted with 10 % acetic acid and OD was measured at 560 nm.
BrdU incorporation and apoptosis assays
Cells were labelled with BrdU for 1 h and analysed by fluorescence-activated cell sorting (FACS). For detection of apoptosis, cells were detached with trypsin and stained with Annexin V-pacific blue and propidium iodide (PrI). Relative proportion of viable cells and cells in early or late apoptosis were determined by FACS analysis.
Oxygen consumption rates
Experiments were performed in a 96-well format using a SeahorseBioscience XF96 Extracellular Flux Analyser (Software Version 1.4) in assay medium supplemented with 1 mM sodium pyruvate and 10 mM glucose, with pH adjusted to 7.4. During the experiment, 1.264 μM oligomycin A (Sigma), 0.4 μM FCCP (Sigma) and 1 μM rotenone (Sigma) were injected. Oxygen consumption rates (OCR) were normalised to cell number.
Spheroid growth assays
Cells were mixed with 2 % matrigel (BD Biosciences 356231) in culture medium and placed in 96-well ultralow attachment plates (Costar). Spheroid formation was initiated by centrifugation at 850×g for 10 min. Fresh growth medium was administered every 72 h. Spheroid size was determined by automated imaging on an inverted microscope (Axiovert 100 M, Carl Zeiss). Spheroids were fixed in neutral buffered formalin (NBF), suspended in 2 % agarose and paraffin embedded. Sections were incubated twice for 3 min in xylene, twice for 3 min in 100 % ethanol, twice for 2 min in 95 % industrial methylated spirit (IMS), twice for 2 min in 70 % IMS and rehydrated for 2 min in 50 % IMS before immunohistochemical analysis.
Tissue microarray (TMA) slides (BR1921a) were purchased from US Biomax, Inc. (Rockville, MD, USA). Antibody staining was performed as in . Legal consent was obtained by the company prior to collection of material.
Comparative expression and survival analysis
Comparative expression analysis was performed using Oncomine (Compendia Bioscience, Thermo Fisher Scientific, Grand Island, NY, USA). Correlations between SCD expression and relapse-free survival in breast cancer were calculated using the GOBO analysis tool .
Prostate orthograft tumour model
All the animal experiments conducted for this study were carried out with ethical approval from University of Glasgow under the revised Animal (Scientific Procedures) Act 1986 and the EU Directive 2010/63/EU (PPL 30/3185). Animals were housed in individual ventilated cages in a barrier facility proactive in environmental enrichment. Balb/C-nude male mice were obtained from Charles River Research Models and Services (UK). A midline lower abdominal incision was made on mice anesthetized by isoflurane inhalation. Using a 1-cc syringe with a 27-gauge needle, 2 × 106 cells in 50 μl of serum-free phenol red-free glutamine containing RPMI were injected in one of the anterior prostate lobes. Mice were gavaged daily (0.2 ml) with doxycycline (10 mg/ml) starting at 10 days post-surgery or as indicated. Mice were anaesthetised, intraperitoneally injected with VivoGlo™ Luciferin (Promega) and imaged using an IVIS Spectrum imaging system. Images were analysed using the IVIS Living Image software. Ultrasound imaging was carried out in three-dimensional (3D)-mode using Vevo® 3100 Imaging System (Visual Sonics).
Targeted RNAi screen identifies SCD as an essential gene in breast and prostate cancer cells
To confirm the reduction in cell number observed after SCD silencing is due to loss of enzyme activity, we used two structurally unrelated SCD inhibitors: 4-(2-chlorophenoxy)-N-(3-(3-methylcarbamoyl)phenyl)piperidine-1-carboxamide (SCDi I) and 3-[4-(2-chloro-5-fluorophenoxy)-1-piperidinyl]-6-(5-methyl-1,3,4-oxadiazol-2-yl)-pyridazine (SCDi II). Treatment with SCDi I reduced proliferation of breast and prostate cancer cell lines grown in low serum in a dose-dependent manner, while having almost no effect in full serum (Fig. 1d), thus phenocopying the effect of gene silencing. Similar results were obtained with SCDi II in DU145 cells (Fig. 1e). Moreover, silencing of SCD using two different shRNA hairpins also efficiently reduced cell numbers of DU145 cells grown in low serum (Additional file 1: Figure S1b and c). Together, these results implicate SCD in growth and survival of cancer cells under serum-deprived conditions.
SCD expression is high in human prostate and breast cancers and correlates with tumour grade
Low serum conditions alter the cellular lipid profile of cancer cells
We next examined the composition of intracellular-free FAs (FFAs) in DU145 cells grown under full and low serum conditions. Strikingly, cells displayed high amounts and relative abundance of mono-unsaturated FFAs (Fig. 3b and Additional file 2: Figure S2a). In particular, the ratio between oleic acid (18:1) and stearic acid (18:0) was significantly higher in the intracellular FFA pool compared to serum FFAs (Additional file 2: Figure S2b), suggesting that mono-unsaturated FFAs are not derived from the serum FFA pool. However, it is also possible that a proportion of intracellular mono-unsaturated FFAs are derived from desaturated phospholipids, such as lysophosphatidylcholine, as demonstrated previously . Interestingly, the levels of mono-unsaturated FFAs were further increased when cells were cultured in low serum (Fig. 3b), suggesting that the high abundance of unsaturated intracellular FFAs in cancer cells is a result of intrinsic desaturase activity and is increased under conditions when extracellular lipid supply is reduced and de novo FA synthesis is activated.
SCD maintains fatty acid desaturation and viability in cancer cells
We next investigated the effect of restoring levels of mono-unsaturated FFA on cellular lipid pools by providing cells with exogenous oleic acid at the same concentration found in full medium. Efficient coupling of oleic acid to BSA was confirmed by mass spectrometry (Additional file 2: Figure S2c). Interestingly, addition of oleic acid fully restored the amounts of mono- and poly-unsaturated FFAs in the cells (Fig. 4a and Additional file 3: Figure S3b) and reduced the relative proportion of saturated FFAs in the cellular FFA pool (Additional file 3: Figure S3d).
We also analysed acyl-chain length and composition of membrane phosphoglycerides in cancer cells cultured in full or low serum. Lipid species containing mono-unsaturated acyl-chains showed a marked increase in the amount in cells cultured in low serum (Additional file 4: Figure S4), suggesting that newly synthesised FAs contribute to the phosphoglyceride pool under those conditions. Inhibition of SCD caused a marked reduction in phosphatidylethanolamine (PE) containing mono-unsaturated acyl-chains, with the major two species, 36:1-PE and 36:2-PE, showing more than 50 % reductions (Additional file 5: Figure S5a and b). This resulted in a relative increase of long-chain poly-unsaturated PE species to the total pool (Additional file 5: Figure S5c). Interestingly, re-addition of oleic acid restored levels of mono-unsaturated PE (Additional file 5: Figure S5b), while reducing the relative proportion of some poly-unsaturated species (Additional file 5: Figure S5c). Similar results were also obtained for phosphatidylinositol (PI) and phosphatidylserine (PS) (Additional file 6: Figure S6 and Additional file 7: Figure S7, respectively).
To confirm that the loss in cell number after treatment with siRNA and SCDi against SCD was indeed due to reduced availability of mono-unsaturated FAs, we reasoned that supplementing cancer cells with oleic acid, the enzymatic product of the SCD reaction, would rescue cell survival. Indeed, supplementation with BSA-oleate completely restored cell viability in the presence of SCD inhibitor (Fig. 4b) and prevented the reduction in cell number following siRNA-mediated SCD silencing (Fig. 4c). Importantly, addition of BSA-oleate did not affect the viability of cells after silencing of PLK1, confirming the specificity of the assay (Fig. 4c). Moreover, supplementation of oleic acid not only provided immediate relief from SCD inhibition but also supported long-term maintenance of viable cells in the presence of the SCD inhibitor in low serum conditions (Fig. 4d). This suggests that maintaining a pool of mono-unsaturated FAs is essential for sustained cancer cell viability.
Inhibition of SCD alters cardiolipin composition and sensitises cancer cells towards apoptosis
We therefore investigated whether modulation of FA availability from serum alters composition of CL molecules in cancer cells. The major CL species in DU145 prostate cancer cells grown in full serum contain mainly shorter acyl-chains with one or two double bonds each (Additional file 8: Figure S8a). Cells cultured in low serum contain significantly higher levels of mono- and poly-unsaturated CL species (Fig. 5c), most prominently 70:4-CL and 72:4-CL (Additional file 8: Figure S8a), indicating that reduced availability of exogenous lipids and increased de novo lipid synthesis also affects the CL pool. Moreover, inhibition of SCD caused a substantial decrease in the levels of mono-unsaturated CL species, while poly-unsaturated CLs, particularly those with longer acyl-chains, were not affected (Fig. 5d and Additional file 8: Figure S8b). Importantly, addition of oleic acid restored the levels of mono-unsaturated CL species (Fig. 5d).
As mentioned above, CLs are integral components of mitochondrial inner membranes and control the release of cytochrome C during apoptosis . Indeed, reduced CL synthesis in neonatal cardiomyocytes exposed to palmitate has been shown to cause the release of cytochrome C leading to apoptosis , indicating that CL composition not only modulates the cellular response to apoptotic stimuli but can also initiate cell death. We therefore investigated the effect of SCD inhibition on cytochrome C release and apoptosis in DU145 cells. Treatment with SCD inhibitor caused the efficient release of cytochrome C into the cytoplasm in cells grown in low serum (Fig. 5e). This was completely blocked by supplementing the cells with exogenous oleic acid (Fig. 5e), confirming that the release of cytochrome C is caused by a lack of mono-unsaturated FAs. In contrast to some previous reports [26–28], SCD inhibition did not alter Akt phosphorylation, presumably because the cells were cultured under reduced serum conditions. Instead, we noticed that treatment with oleic acid induced a small increase in Akt phosphorylation (Fig. 5e). However, pre-treatment with an Akt inhibitor did not block the ability of oleic acid to prevent cytochrome C release (Fig. 5f), indicating that oleic acid supports cell survival by restoring cellular lipid composition rather than altering Akt signalling. Moreover, induction of PARP cleavage in response to SCD inhibition or silencing was also completely blocked by oleic acid (Fig. 5g, h).
As cytochrome C is an integral component of the electron transport chain (ETC.), we also analysed the effect of SCD inhibition on oxygen consumption in cancer cells. Treatment of DU145 or MDA-MB-468 cells with SCD inhibitor in low serum not only reduced the basal OCR but also lowered maximal respiration in the uncoupled state, confirming that the activity of the ETC. is impaired in these cells (Fig. 5i). Moreover, SCD inhibition sensitised cancer cells towards metformin and rotenone, two inhibitors of complex I of the respiratory chain (Fig. 5j, k). We also investigated whether attenuation of SCD activity also increases the sensitivity of cancer cells towards drugs that induce apoptosis via the mitochondrial death pathway. While treatment with low doses of SCD inhibitor alone had little effect on cell viability, we found that it enhanced the toxicity of paclitaxel and staurosporine (Fig. 5l, m). Taken together, these results suggest that SCD inhibition causes alterations in the composition of lipids that are crucial for the function of the inner mitochondrial membrane and that altered lipid desaturation can enhance the susceptibility of cancer cells towards chemotherapeutic drugs and metabolic inhibitors.
Spheroid cultures exhibit tumour-like lipid composition and depend on SCD activity
Cancer cells can be grown as multi-layered 3D spheroids. These structures recreate the nutrient and oxygen gradients that cancer cells are exposed to within a growing tumour. We found that T47D cells grown as tumour spheroids show a similar increase in poly-unsaturated DG species as found in tumours (Fig. 6b and Additional file 9: Figure S9c). Moreover, increased levels of several mono-unsaturated lipid species, most prominently PI and lysophosphatidic acid (LPA), were also detected (Fig. 6c). To examine the effect of SCD ablation on lipid composition in these conditions, we used DU145 cells expressing inducible shRNA hairpins targeting SCD to generate spheroids. These were cultured in full medium, and gene silencing was activated with doxycycline. As can be seen from the data shown in Fig. 6d, depletion of SCD caused a marked decrease in the relative amount of mono- and poly-unsaturated FFA species (Fig. 6d and Additional file 10: Figure S10a). Furthermore, the poly-unsaturated DG population, which was selectively upregulated in tumours (see Fig. 6a), was reduced when SCD was ablated (Fig. 6e and Additional file 10: Figure S10b). This was accompanied by a substantial reduction in spheroid size despite the presence of serum in the culture medium (Fig. 6f). A similar reduction on spheroid size was observed in MDA-MB-468 breast cancer cells following inhibition of SCD activity (Fig. 6g). Interestingly, this activity was seen at a concentration of the drug that was ineffective at inhibiting cell growth in 2D cultures in full serum (see Fig. 1d), suggesting that spheroid culture sensitises cancer cells towards SCD inhibition. These data confirm that SCD activity is essential to maintain levels of mono-unsaturated FAs in cancer cells exposed to the compromised metabolic environment created by 3D culture and that these FAs are rate limiting for cell growth under these conditions.
SCD is essential for orthotopic growth of prostate cancers
Having shown that SCD is important to control lipid provision under conditions that resemble the metabolic microenvironment of tumours, we next investigated whether SCD expression is also essential for tumour growth. We implanted DU145 prostate cancer cells expressing inducible shRNAs targeting SCD orthotopically into the prostates of immunocompromised mice (nu/nu). Induction of gene silencing was induced with doxycycline. In one cohort, treatment was started 10 days after implantation when tumours were already detectable by bioluminescence imaging (early). In order to investigate the effect of SCD inhibition on established tumours, a second cohort was treated with doxycycline starting at day 47 post-implantation (late). Tumour growth was monitored over time, and mice were culled upon reaching the humane endpoint.
In summary, our results demonstrate that SCD is an important enzyme to support the enhanced de novo lipid synthesis and desaturation in cancer cells under conditions where exogenous lipids are limiting. SCD controls the production of lipid species that are important for cell viability and determine the sensitivity of cancer cells towards chemotherapeutic agents. Finally, SCD is essential for tumour cell growth within the metabolic restraints of the tissue that they arise in.
Increased lipid biosynthesis is an important feature of cancer cells [6, 7, 29]. Inhibitors of fatty acid synthase (FASN) have so far shown limited therapeutic promise due to systemic toxicity . Defining the exact contribution of lipid metabolism enzymes to cancer cell growth and survival is crucial to identify potential new therapeutic targets.
In this study, we analysed the response of breast and prostate cancer cell lines to the depletion of SCD, a rate-limiting enzyme for the production of mono-unsaturated fatty acids. We found that silencing of SCD reduced proliferation in almost all cancer cells lines studied without affecting the viability of non-malignant epithelial cell lines derived from the same tissues. In contrast to previous reports , we did not observe a cell cycle arrest in response to SCD inhibition. This could be because the cells were already cycling slower due to the reduced availability of serum-derived growth factors. Nevertheless, the strong and selective dependence of cancer cells on lipid desaturation observed here is in agreement with previous reports [31, 32]. It is possible that increased lipid desaturation maintains cellular functions that are selectively important in cancer cells. Indeed, lipid desaturation was shown to prevent engagement of the unfolded protein response in cancer cells due to their increased rates of protein synthesis [17, 18, 33].
SCD has been previously linked to cancer cell proliferation and survival [34, 35]. Increased expression of SCD has been found in several cancer types, including prostate [27, 36], liver , kidney  and breast cancer . Inhibition of SCD by expression of antisense RNA reduced tumour formation in human lung adenocarcinoma cancer cells  while pharmacological inhibition of SCD was effective in blocking the growth of gastric and colon cancer cells [32, 36]. SCD also promotes proliferation and disease progression in prostate cancer by affecting cellular signaling cascades and modulating androgen receptor transactivation [27, 40]. Furthermore, it has been shown that breast cancers contain higher proportions of saturated and mono-unsaturated lipid species, indicative of increased lipid synthesis and desaturation rather than uptake of dietary lipids . The same study also found reduced viability after SCD silencing in breast cancer cells , confirming the importance of this enzyme for cancer metabolism. Finally, altered serum lipids also offer diagnostic opportunities as reduced levels of triacylglycerides containing oleic acid were found to be indicative of prolonged survival in breast cancer patients after neoadjuvant treatment .
Our study provides additional evidence for the important role of SCD in cancer. We found that SCD is overexpressed in breast and prostate cancers compared to normal tissues. In breast cancer, high levels of SCD expression were confined to invasive ductal breast carcinomas but absent in invasive lobular carcinomas, providing important subtype specification. Moreover, SCD expression correlated with tumour grade and was indicative of disease relapse. SCD expression also determined reduced progression-free survival of high-grade tumours and those stratified by the PAM50 gene expression signature, which defines basal-like aggressive disease that presently has limited therapeutic options . In keeping with this finding, three out of the four triple negative breast cancer cell lines used in this study showed high sensitivity towards SCD depletion.
Our study further demonstrates that the dependence of cancer cells on SCD is strongly determined by the availability of exogenous lipids. Lipid deprivation increased de novo lipid biosynthesis and enhanced the contribution of mono-unsaturated FAs to the cellular lipid pool. We also confirmed that the effect of SCD silencing on cancer cell viability was due to inhibition of enzymatic activity, as two structurally unrelated inhibitors of SCD activity showed comparable efficacy in reducing proliferation and survival in cancer cells. Both inhibitors reduced the levels of mono- and poly-unsaturated FA species and increased the relative abundance of saturated forms. This shift towards higher saturation could also be observed in the acyl chains of several membrane phosphoglycerides, indicating that SCD inhibition alters the composition of important cellular lipid species. However, the amounts of some phosphoglyceride species containing long-chain poly-unsaturated FAs were increased following SCD inhibition, suggesting that cells can remodel existing lipid pools or selectively take up certain lipid species when desaturation is blocked. Indeed, it has been shown that the selective uptake of unsaturated lyso-phospholipids is induced by hypoxia and in Ras-transformed cells .
Interestingly, we found that re-addition of oleic acid restored most of the alterations in lipid composition induced by SCD inhibition, confirming that this mono-unsaturated FA is indeed rate-limiting for the generation of a large part of the cellular lipid spectrum when exogenous lipids are scarce. Addition of oleic acid also prevented the loss of cell viability in response to SCD silencing or inhibition. This effect was maintained over several days, providing sufficient lipid substrate for membrane biosynthesis during this time. Of particular importance in this context is the continuous functionality of the inner mitochondrial membrane, which contains a high proportion of cardiolipins (CL). The degree of desaturation determines the function of this highly specialized lipid class, and the acyl chains in CL molecules undergo constant remodeling . The composition of different CL molecules within the inner mitochondrial membrane can modulate the activity of ETC. complexes . Moreover, the acyl chains of CL molecules interact with cytochrome c, thereby affecting both respiration and apoptosis .
We found that inhibition of SCD reduced levels of mono-unsaturated CL species, with a specific reduction in mono-unsaturated species. This was accompanied by the release of cytochrome C and induction of apoptosis. Sub-lethal doses of SCD inhibitor also increased the sensitivity of cancer cells towards chemotherapeutic agents and inhibitors of mitochondrial respiratory complexes. This is in agreement with a recent study showing that betulinic acid alters the saturation of CL species, causing mitochondrial damage and cytochrome C release, most likely through inhibition of SCD function . It is also possible that the reported activation of AMP-activated protein kinase (AMPK) by SCD  could involve inhibition of mitochondrial activity by altering cellular CL composition.
We also observed that different culture conditions have substantial effects on lipid composition. Human cancer cells grown as orthotopic tumours exhibited specific lipid profiles that were characterised by high levels of mono- and poly-unsaturated lipid species. These lipid profiles were also detected in tumour spheroids, an experimental system that recreates the oxygen and nutrient gradients observed in tumors. Silencing of SCD reduced the ability of cancer cells to grow as spheroids, indicating that these conditions restrict the access to exogenous lipids making cancer cells dependent on SCD function. Cells within metabolically restricted hypoxic areas of human tumors frequently show therapy resistance and increased stem cell capabilities , making the selective targeting of this niche a therapeutic priority.
Most importantly, we found that depletion of SCD efficiently blocked the ability of prostate cancer cells to grow as orthotopic tumour xenografts, resulting in reduced tumour volume and prolonged survival of the host. This effect was also observed when SCD silencing was initiated after the tumours had already grown to a substantial size, indicating that targeting SCD could offer treatment opportunities in established cancer. It should be noted that the RNAi strategy used here selectively targets SCD only in cancer cells. This excludes potential effects on desaturase activity in cells of the tumour stroma or global alterations in the lipid metabolism of the host, which can contribute to tumour inhibition by systemic SCD inhibition.
This study demonstrates that SCD is deregulated in human breast and prostate cancers and essential for cancer cell survival and tumour growth. Inhibition of SCD altered cellular lipid composition, leading to a distinct reduction in particular lipid species that depend on the availability of mono-unsaturated FAs. This phenotype was only observed under conditions when exogenous lipid sources were limited. The functional consequences of deregulated lipid metabolism in response to SCD inhibition included mitochondrial dysfunction, release of cytochrome C and induction of apoptosis, confirming the important role of fatty acid desaturation for essential cellular processes.
Taken together, our results highlight the importance of SCD for lipid provision in cancer cells under the metabolically compromised conditions that are likely to be encountered within the tumour microenvironment (Fig. 7e). We conclude that synthesis of mono-unsaturated FAs by SCD represents a metabolic bottleneck of lipid biosynthesis and thus provides a suitable target for therapeutic development.
The authors would like to thank Neil Jones and Angus Lauder from Cancer Research Technology for the management of the consortium and scientific advice.
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