Cancer cell lipid class homeostasis is altered under nutrient-deprivation but stable under hypoxia

Under oxygen/nutrient deprivation cancer cells modify the balance between fatty acid (FA) synthesis and uptake, which alters the levels of individual triglyceride or phospholipid sub-species. These modifications may affect survival and drug-uptake in cancer cells. Here, we aimed to attain a more holistic overview of the lipidomic profiles of cancer cells under stress and assess the changes in major lipid-classes. First, expressions of markers of FA synthesis/uptake in cancer cells were assessed and found to be differentially regulated under metabolic stress. Next, we performed a broad lipidomics assay, comprising 244 lipids from six major classes, which allowed us to investigate robust stress induced changes in median levels of different lipid classes -additionally stratified by fatty acid side chain saturation status. The lipidomic profiles of cancer cells were predominantly affected by nutrient-deprivation. Neutral lipid compositions were markedly modified under serum-deprivation and, strikingly, the cellular level of triglyceride subspecies decreased with increasing number of double bonds in their fatty acyl chains. In contrast, cancer cells maintained lipid class homeostasis under hypoxic stress. We conclude that although the levels of individual lipid moieties alter under hypoxia, the robust averages of broader lipid class remain unchanged.

Lipid metabolism has emerged as an important aspect of cancer cell metabolism and is widely shown to be associated with various malignant processes (1-3). Cancer cells require a constant supply of lipids for membrane biogenesis and protein modifications. Several studies have shown that, in order to cope with these increased demands, cancer cells activate de novo lipid synthesis pathways (4-8). Fatty acid synthase (FASN) -a key-regulator of de novo fatty acid (FA) synthesis-has been extensively shown to fuel cancer cell proliferation and malignant progression (3). Expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) -the rate-controlling enzyme of the mevalonate pathway-is also up-regulated in cancers (9). Importantly, inhibition of FA synthesis or cholesterol synthesis pathways results in growtharrest of lipogenic tumor cells rendering these pathways interesting targets for antineoplastic therapy (4, 10-16). Although endogenous FA synthesis has historically been considered the principal source of fatty acids (FAs) in cancer cells, lipolytic phenotypes are also widely recognized (reviewed in (17)). For example, it has been reported that in addition to the markers of de novo synthesis (FASN) different cancer cells also express markers of lipolysis (lipoprotein lipase, LPL) and exogenous FA uptake (CD36) (18).
Additional support for coordinated lipolytic and lipogenic metabolism in cancer cells involves the incorporation of endogenously synthesized FAs into cellular neutral lipid stores. Nomura et al. (19) have proposed that complementary lipolytic pathways are required to release fatty acyl moieties from these lipid reservoirs and have demonstrated a specific role for an intracellular lipase, monoacyl glycerol lipase (MGLL), in promoting tumorigenesis. MGLL provides, by de-esterification, a stream of intracellular free FAs to fuel proliferation, growth, and migration. Taken together, these findings are compatible with the notion that both lipogenesis and lipolysis may be utilized by cancer cells to fulfill their FA requirements.
The mode of FA acquisition -via de novo synthesis or uptake-may significantly affect the lipidomic profiles of cancer cells. Mammalian cells have a limited ability to synthesize polyunsaturated fatty acids de novo, as they lack the Δ lipid peroxidation than polyunsaturated acyl chains, de novo synthesis was proposed to make cancer cells more resistant to oxidative stress-induced cell death (20). Moreover, as saturated lipids pack more densely their increased levels alter lateral and transverse membrane dynamics that may limit the uptake of drugs, making the cancer cells more resistant to therapy (20). Hence, the balance between FA synthesis and uptake may have important therapeutic implications.
Cancer cells are shown to modify balances of FA synthesis and uptake under metabolic stress, i.e.
induced by oxygen and nutrient deprivation (3,21). This metabolic flexibility is particularly important for cancer cells within solid tumors that are exposed to oxygen-and nutrient-gradients depending on their distance from the nearest blood vessels. The inefficient vascularization limits access of various nutrientssuch as amino acids, sugars and lipids-to tumor tissues. The effect of oxygen/nutrient deprivation on leukemic cells was presumed to be inconsequential and remained long overlooked. This view is also being revised and hypoxia has been shown to influence leukemic cell proliferation, differentiation, and resistance to chemotherapy (reviewed in (22)). It will also be interesting to study the impact of oxygen/nutrient deprivation on metabolic pathways in leukemic cells.
To date, the effect of metabolic stress on lipid metabolism in cancer cells appears inconsistent. shown that under hypoxia breast cancer cells display modified phospholipid profiles mainly characterized by the presence of shorter and more saturated acyl chains while other lipid classes were not considered (27). Another study reported that under hypoxic conditions cellular levels of triglycerides with three double bonds were significantly decreased in MCF7 breast cancer cells (26), but significantly increased in U87 glioblastoma cells (26), but data on membrane lipids were not collected. Nutrient deprivation, specifically low-lipid environment, is also shown to affect de novo lipid synthesis pathways in cancer cells (28,29). It has been reported that cancer cells from different origins differentially activate and thrive on de novo lipid synthesis pathways in a low-lipid environment. It was shown that cancer cells that attain the highest lipogenic activity under lipid-reduced environment are best able to cope with lipid reduction in term of proliferative capacity (28). Hence, the changes in lipid metabolism and lipidomic profiles of cancer cells that are induced by environmental stress appear to be cell-type specific. Together, these data indicate that both genetic background and environmental conditions may determine the relative dependence of cancer cells on de novo lipid synthesis versus lipid uptake.
Herein, to study the complex interplay between metabolic stress and lipid metabolism in cancer cells, we selected a biologically diverse panel of cancer cell lines -three leukemia cell lines (to cover intra-group variance), two colon cancer cell lines and one lung cancer cell line. We were mainly interested in studying the impact of physiologically relevant metabolic stress on lipid metabolism in cancer cells. To achieve that cancer cells were cultivated under nutrient-deprivation and hypoxia -that closely mimics the in vivo conditions. The expression of relevant markers was assessed to study the relative dependence of cancer cells on de novo lipid synthesis versus lipid uptake/degradation pathways under metabolic stress.
In order to gain more systematic insight on the effects of metabolic stress on lipidomic profiles we performed a broad lipidomics assay comprising 244 lipids from six major classes. To this end we identified multiple changes in lipidomic profiles of cancer cells cultivated under low-serum or lipiddeficient conditions. Under hypoxic stress cancer cells displayed alterations in cell proliferation rates and expression profiles of various lipid metabolism associated genes. Interestingly, no robust changes were observed in lipidomic profiles of hypoxic cancer cells indicating that the cells maintain lipid class homeostasis.

Lipid Extractions
First, the cell pellets were washed with 0.5 mL 0.9% NaCl. For extraction of lipids the pellets were resuspended in 1 ml ice-cold MMC (1:1:1 v/v/v methanol/MTBE/chloroform). Samples were incubated on an ultrasonic bath for two minutes. Phase separation was induced by adding 300 μ L MS-grade water.
After 10 min incubation, the samples were centrifuged for 10 min at 1000 rpm and the upper (organic) phase was collected. Then 200 μ L of collected organic phase were dried in a vacuum rotator and stored at -20 °C until analysis.

Lipidomic Profiling
Dried sample extracts were reconstituted in 100 µL 2:1:1 v/v/v isopropanol/acetonitrile/water. 5 µL aliquots were injected into an ACQUITY I-class ultra-performance liquid chromatography (UPLC) system (Waters, Germany) coupled to an Impact II high-resolution quadrupole time-of-flight mass spectrometer (Bruker Daltonik GmbH, Germany Identification score cut off (60%). Identified peaks were exported to a text file and subjected to statistical analysis.

Statistical analysis
The differences between groups were analyzed by ANOVA or t-test (paired or unpaired), where applicable. Statistical analyses and graphical representations for lipidomic data and quantitative RT-PCR data were performed using the R software environment 3.4.2 (http://cran.r-project.org/) or MetaboAnalyst 3.5 (http://www.metaboanalyst.ca/faces/home.xhtml). P-values <0.05 were considered statistically significant and indicated when different. The expression of lipolytic markers (LPL and MGLL) was also assessed for the selected cell line panel.

Comparison of baseline expression of selected markers for
We observed consistent expression of LPL only in SW480 and KU812. The expression of MGLL was strikingly different among the selected cell lines with up to ~250-folds difference between SW620 and A549 (Figure 1a). Expression of CD36 -fatty acid uptake marker-was only detectable in KU812. These data indicate that the selected cell lines are significantly different in terms of baseline levels of lipid metabolism related transcripts.

Comparison of baseline lipidomic profiles of cancer cell lines
Next, we compared the baseline lipidomic profiles of the selected cell lines. To integrate the lipidomics data with gene expression profiles we determined the lipidomic profiles of the same samples that were used for qPCR analysis. Global lipidomic profiling using Liquid Chromatography-Mass Spectrometry (LC-MS) followed by automatic annotation using ) allowed to detect 244 lipid compounds each present in at least 90% of all samples ( Table 1)

Effect of metabolic stress on cell proliferation and expression of different lipid metabolism-related genes in cancer cells
We have previously shown that cancer cells cultivated under low-lipid conditions display differential growth patterns (28). Here, we first studied the effect of different metabolically-stressed culture conditions on cell proliferation in various cancer cells. To induce metabolic stress, cells were cultivated for 48h under following conditions: lipoprotein deficient medium (LPDS serum), low-serum medium (2% serum), hypoxia (2% O2), or hypoxia in combination with low-serum medium. Figure 3 shows proliferation rates of cancer cells cultivated under different cell culture conditions. Here, for each cell line the data were normalized to its proliferation rate under normal condition. Thereby, we remove the existing differences in baseline proliferation rates of selected cell lines (Supplementary Figure 2) and emphasize on stress-induced changes in proliferation rates. We observed that metabolic stress significantly impacted proliferation rates of most cell lines (Figure 3). KU812, SW480 and SW620 showed decreased proliferation rates when cultivated in media containing lipoprotein-deficient serum (LPDS). KU812 and SW480 express LPL and CD36 proteins that are involved in lipolysis and uptake of extracellular fatty acids. Therefore, they might be more sensitive to LPDS medium. All cell lines except SW620 showed reduced proliferation rates under low-serum environment. Hypoxic conditions induced decreased proliferation rates in A549 and SW480. When cultivated under hypoxia in combination with low-serum medium all cell lines except SW620 displayed reduced proliferation rates.
It has been reported that cancer cell lines differentially regulate de novo lipid synthesis pathways under oxygen and/or nutrient deprivation (23-25, 28). Here, we also observed differential impact of metabolic stress on expression of FASN and HMGCR. This data also indicates cell-type specific regulation of de novo lipid synthesis pathways under metabolic stress (Supplementary Figure 3). mRNA levels of FASN and HMGCR under stress conditions also displayed highly positive correlation (R 2 =0.75; P <0.0001) (Supplementary Figure 4). The expression of markers of lipid uptake/degradation (LPL, MGLL and CD36) was also differentially affected by metabolic stress (Supplementary Figure 3).

Effect of metabolic stress on lipidomic profile of cancer cells
Next, we studied the effects of metabolic stress on lipidomic profiles of cancer cells. Previous studies have already shown various stress-related effects on individual lipid-moieties. Our broad lipidomic assay allowed us to assess the impact of metabolic stress on robust averages of broader lipid-classes, providing a more holistic overview of cancer cell lipidomic profiles.

Discussion
The presented work aimed to determine the impact of metabolic stress on expression of selected lipid metabolism genes, lipidomic profiles and cell proliferation in biologically diverse cancer cells. We observed that under metabolic stress different cancer cell lines showed differential expression of markers for de novo lipid synthesis and lipid-uptake/degradation. These data confirm previous works that showed cell-type specific regulation of lipid metabolism under oxygen and nutrient-deprivation (28) a  n  c  h  e  z  -C  a  b  a  l  l  e  r  o  ,  E  .  N  i  n  t  o  u  ,  V  .  G  .  B  o  i  a  d  j  i  e  v  a  ,  F  .  P  i  c  a  t  o  s  t  e  ,  A  .  G  u  b  e  r  n  ,  a  n  d  E  .  C  l  a  r  o  .  2  0  1  3  .  C  e  l  l  s  u  r  v  i  v  a  l  d  u  r  i  n  g  c  o  m  p  l  e  t  e  n  u  t  r  i  e  n  t  d  e  p  r  i  v  a  t  i  o  n  d  e  p  e  n  d  s  o  n  l  i  p  i  d  d  r  o  p  l  e  t  -f  u  e  l  e  d  b  e  t  a  o  x  i  d  a  t  i  o  n  o  f  f  a  t  t  y  a  c  i  d  s  .   J  B  i  o  l  C  h  e  m   2  8  8  :  2  7  7  7  7  -2  7  7  8  8  .   4  1  .  H  a  p  a  l  a  ,  I  .  ,  E  .  M  a  r  z  a  ,  a  n  d  T  .  F  e  r  r  e  i  r  a  .  2  0  1  1  .  I  s  f  a  t  s  o  b  a  d  ?  M  o  d  u  l  a  t  i  o  n  o  f  e  n  d  o  p  l  a  s  m  i  c  r  e  t  i  c  u  l  u     LS+Hyp stress was not tested for SW480 and SW620 cell lines.
5 lar on ak ng ed.