Cancer Cells Differentially Activate and Thrive on <i>De Novo</i> Lipid Synthesis Pathways in a Low-Lipid Environment

<div><p>Increased lipogenesis is a hallmark of a wide variety of cancers and is under intense investigation as potential antineoplastic target. Although brisk lipogenesis is observed in the presence of exogenous lipids, evidence is mounting that these lipids may adversely affect the efficacy of inhibitors of lipogenic pathways. Therefore, to fully exploit the therapeutic potential of lipid synthesis inhibitors, a better understanding of the interrelationship between <i>de novo</i> lipid synthesis and exogenous lipids and their respective role in cancer cell proliferation and therapeutic response to lipogenesis inhibitors is of critical importance. Here, we show that the proliferation of various cancer cell lines (PC3M, HepG2, HOP62 and T24) is attenuated when cultured in lipid-reduced conditions in a cell line-dependent manner, with PC3M being the least affected. Interestingly, all cell lines - lipogenic (PC3M, HepG2, HOP62) as well as non-lipogenic (T24) - raised their lipogenic activity in these conditions, albeit to a different degree. Cells that attained the highest lipogenic activity under these conditions were best able to cope with lipid reduction in term of proliferative capacity. Supplementation of the medium with very low density lipoproteins, free fatty acids and cholesterol reversed this activation, indicating that the mere lack of lipids is sufficient to activate <i>de novo</i> lipogenesis in cancer cells. Consequently, cancer cells grown in lipid-reduced conditions became more dependent on <i>de novo</i> lipid synthesis pathways and were more sensitive to inhibitors of lipogenic pathways, like Soraphen A and Simvastatin. Collectively, these data indicate that limitation of access to exogenous lipids, as may occur in intact tumors, activates <i>de novo</i> lipogenesis is cancer cells, helps them to thrive under these conditions and makes them more vulnerable to lipogenesis inhibitors. These observations have important implications for the design of new antineoplastic strategies targeting the cancer cell's lipid metabolism.</p></div

Lipid-reduced (LR) growth conditions attenuate 2D proliferation rate of HOP62, HepG2 and T24 cells but not of PC3M cells.

<p><b>(a)</b> Proliferation curves for PC3M, HOP62, HepG2 and T24 cells. Cells were seeded and cultivated in normal or lipid-reduced medium and cell proliferation was monitored by <i>Incucyte real-time imaging.</i> The panels on the right side of each proliferation graph show the phase contrast image of the corresponding cell line in both conditions. Results are representative of three independent experiments</p><p>. <b>(b)</b> The number of live cells was counted using a trypan blue dye exclusion method, after 72 hours of culturing in normal (N) or LR medium. *Significantly different (*p≤0,05; **p≤0,01; ***p≤0,001), n.s. not significant (p>0,05).</p><p></p

Lipid-reduced (LR) medium conditions increase the activation of SREBP1 and SREBP2 in some but not all cancer cell lines.

<p><b>(a)</b> Representative virtual blot of Simple Western analysis of precursor and active SREBP1 expression in HepG2, PC3M, HOP62 and T24 cells after 72 hours cultivation in normal (N) or LR medium conditions. Alpha-tubulin is used as a loading control. Different exposures of precursor and active SREBP1 are shown in order to have an accurate exposure for both forms. For original data see Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106913#pone.0106913.s003" target="_blank">Figure S3a</a>-d. <b>(b)</b> Quantitative analysis of Simple Western. data, expressed as area under the curve (AUC). Expression of SREBP1 was corrected for the loading control alpha-tubulin. Graph represents mean ± S.D. (n = 2–3). <b>(c)</b> Representative virtual blot of Simple Western analysis of precursor and active SREBP2 expression in HepG2, PC3M, HOP62 and T24 cells after 72 hours cultivation in normal (N) or LR medium conditions. Alpha-tubulin is used as a loading control. Different exposures of precursor and active SREBP2 are shown in order to have an accurate exposure for both forms. For original data see Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106913#pone.0106913.s003" target="_blank">Figure S3e</a>-h. <b>(d)</b> Quantificative analysis of Simple Western. data, expressed as area under the curve (AUC). Expression of SREBP2 was corrected for the loading control alpha-tubulin. Graph represents mean ± S.D. (n = 2–3).</p

Lipid-reduced (LR) growth conditions differentially increase expression of FASN, HMGCR, ACLY and ACSS2 in different cancer cell lines.

<p>Gene expression of FASN, ACLY, ACSS2 and HMGCR was analyzed by qPCR analysis in <b>(a)</b> HOP62 <b>(b)</b> HepG2 <b>(c)</b> PC3M <b>(d)</b> T24 cells. Cells were cultivated in normal or LR medium for 48 hours. Data are expressed as mean ± S.D of triplicate samples, normalized to TFRC for HOP62, HepG2 and PC3M or to 18S for T24. *Significantly different (*p≤0,05; **p≤0,01; ***p≤0,001), n.s. not significant (p>0,05). <b>(e)</b> FASN and ACLY expression at protein level was analyzed by Western blot analysis in HOP62, HepG2, PC3M and T24 cells cultivated under normal (N) or LR medium for 72 hours. Beta-actin was used as a loading control.</p

Addition of very-low density lipoproteins (VLDL), fatty acids and cholesterol to lipid-reduced (LR) growth conditions reverses the increased activation of the lipogenic pathway.

<p>Gene expression of FASN, ACLY, HMGCR and ACSS2 was analyzed by qPCR in T24 cells were cultured for 48 hours in normal (N) or LR growth conditions in the presence or absence of VLDL <b>(a)</b>, different fatty acid mixtures <b>(b)</b> and different concentrations cholesterol <b>(c)</b>. VLDL was added at a concentration of 607 µg triglycerides/ml serum (corresponding to the concentration triglycerides in normal FBS). Fatty acid (FA) mixtures were as follows, FA Mix 1: 20 µM linoleic (18∶2), 20 µM α-linolenic (18∶3), 5 µM arachidonic (20∶4), 5 µM docosahexaenoic acid (22∶6), FA Mix 2: 10 µM 18∶2, 15 µM 18∶3, 10 µM 20∶4, 15 µM 22∶6 and FA Mix 3: 20 µM 18∶2, 20 µM 18∶3, 5 µM 20∶4, 5 µM 22∶6, 30 µM oleic acid, 30 µM palmitic acid. Different cholesterol (Ch) concentrations are as indicated in the figures (25 µM, 50 µM or 100 µM). Data are normalized to 18S and represented as mean ± S.D. (triplicate per experiment and n = 3). Significance was determined by one-way ANOVA followed by Tukey's multiple comparison test. *Significantly different (*p≤0,05; **p≤0,01; ***p≤0,001; ****p≤0,0001) from normal medium control. ^#Significantly different (^#p≤0,05; ^##p≤0,01; ^###p≤0,001;^####p≤0,0001 ) from LR control. <b>(d, e, f)</b>¹⁴C-incorporation into cellular lipids was determined in T24 cells, cultured for 48 hours in normal (N) or LR growth conditions in the presence or absence of VLDL <b>(d)</b>, different fatty acid mixtures <b>(e)</b> and different concentrations cholesterol <b>(f)</b> as mentioned in (a, b and c). During the last 4 hours ¹⁴C-acetate was added and the incorporation of radioactivity in cellular lipids was normalized to sample DNA content. Representative experiment is shown, experiment was repeated two times. Significance was determined by one-way ANOVA followed by Tukey's multiple comparison test. *Significantly different (*p≤0,05; **p≤0,01; ***p≤0,001; ****p≤0,0001) from normal medium control. ^#Significantly different (^#p≤0,05; ^##p≤0,01; ^###p≤0,001; ^####p≤0,0001) from LR control.</p

Lipid-reduced (LR) growth conditions increase the sensitivity of cancer cells to inhibitors of lipid synthesis pathways.

<p>HepG2, PC3M, HOP62 and T24 cells growing under normal (N) or LR growth conditions were treated with <b>(a)</b> Soraphen A (200 and 500 nM) and <b>(b)</b> Simvastatin (100 or 500 nM). Annexin V and 7-AAD staining was used to detect apoptotic cells after 72 hours of treatment (n = 3). Graph represents mean ± S.D. (n = 2–3). *Significantly different (*p≤0,05; **p≤0,01; ***p≤0,001) from normal growth condition.</p

De novo lipogenesis protects cancer cells from free radicals and chemotherapeutics by promoting membrane lipid saturation.

Activation of de novo lipogenesis in cancer cells is increasingly recognized as a hallmark of aggressive cancers and has been implicated in the production of membranes for rapid cell proliferation. In the current report, we provide evidence that this activation has a more profound role. Using a mass spectrometry-based phospholipid analysis approach, we show that clinical tumor tissues that display the lipogenic phenotype show an increase in the degree of lipid saturation compared with nonlipogenic tumors. Reversal of the lipogenic switch in cancer cells by treatment with the lipogenesis inhibitor soraphen A or by targeting lipogenic enzymes with small interfering RNA leads to a marked decrease in saturated and mono-unsaturated phospholipid species and increases the relative degree of polyunsaturation. Because polyunsaturated acyl chains are more susceptible to peroxidation, inhibition of lipogenesis increases the levels of peroxidation end products and renders cells more susceptible to oxidative stress-induced cell death. As saturated lipids pack more densely, modulation of lipogenesis also alters lateral and transversal membrane dynamics as revealed by diffusion of membrane-targeted green fluorescent protein and by the uptake and response to doxorubicin. These data show that shifting lipid acquisition from lipid uptake toward de novo lipogenesis dramatically changes membrane properties and protects cells from both endogenous and exogenous insults. These findings provide important new insights into the role of de novo lipogenesis in cancer cells, and they provide a rationale for the use of lipogenesis inhibitors as antineoplastic agents and as chemotherapeutic sensitizers