Aberrant lipid metabolism in hepatocellular carcinoma cells as well as immune microenvironment: A review

Abstract Hepatocellular carcinoma (HCC) is a primary malignancy of the liver with a high worldwide prevalence and poor prognosis. Researches are urgently needed on its molecular pathogenesis and biological characteristics. Metabolic reprogramming for adaptation to the tumour microenvironment (TME) has been recognized as a hallmark of cancer. Dysregulation of lipid metabolism especially fatty acid (FA) metabolism, which involved in the alternations of the expression and activity of lipid‐metabolizing enzymes, is a hotspot in recent study, and it may be involved in HCC development and progression. Meanwhile, immune cells are also known as key players in the HCC microenvironment and show complicated crosstalk with cancer cells. Emerging evidence has shown that the functions of immune cells in TME are closely related to abnormal lipid metabolism. In this review, we summarize the recent findings of lipid metabolic reprogramming in TME and relate these findings to HCC progression. Our understanding of dysregulated lipid metabolism and associated signalling pathways may suggest a novel strategy to treat HCC by reprogramming cell lipid metabolism or modulating TME.

which can be hypoxic, acidic and deficient in nutrients, may result in the metabolism of tumour cells and the neighbouring stromal cells, including myeloid cells (such as tumour-associated macrophages, dendritic cells and myeloid-derived suppressor cells) and

lymphocytes (T cells and B cells) for remodelling, thus facilitat-
ing tumour survival, proliferation and metastasis. [2][3][4] The TME of HCC may involve multiple metabolic abnormalities, among which, abnormal lipid metabolism is a fairly new field that attracts wide attention over the past few years. Dysregulation of lipid metabolism, especially for the metabolism of fatty acid (FA) where the aberrantly activated oncogenic signalling pathways alter the lipid-metabolizing enzyme expression and activity, has increasingly been recognized as an important metabolic rewiring phenomenon in tumour cells ( Figure 1) and immunocytes, and it may also participate in HCC development and progression. 3 This review aims to examine the mechanism by which this dysregulation modelled HCC cells and neighbouring immunocytes and support HCC progression and to explore the way for therapeutically targeting the aberrant lipid metabolism to benefit the HCC patients.

| Aberrant lipid metabolism
Increasing evidence suggests that alterations in tumour lipid metabolism, including metabolite abundance and accumulation of lipid metabolic products, lead to tumour development as well as local immunosuppression in the TME. 5 For instance, a previous study illustrated that the deletion of 5-lipoxygenase in the TME promoted lung cancer progression and metastasis through regulating T-cell recruitment. 6 A recent study has analysed the global gene expression profile of HCC, which reveals that genes involved in the biosynthesis of fatty acids (FAs) are universally up-regulated in most HCC tissues compared with the noncancerous liver tissues. 7,8 Typically, FAs function as the signalling molecules, energy sources, and the structural components of cell membrane, all of which are essential for cancer cell proliferation. 9 Normal cells preferentially utilize the circulating exogenous lipids, whereas cancer cells, including HCC cells, show a high de novo lipid synthesis rate, 10 suggesting FA accumulation in tumour cells. The roles of major lipogenic enzymes, such as stearoyl-CoA desaturase (SCD), fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC), have been reported to participate in hepatocarcinogenesis. For instance, the genetic ablation of FASN, which is responsible for synthesizing palmitate (C16:0) from acetyl-CoA and malonyl-CoA in the presence of NADPH, completely suppresses the Akt-driven HCC development through inhibiting the Rictor/mammalian target of rapamycin complex2 (TORC2) signalling. 11 ACC, which converts acetyl-CoA to malonyl-CoA as the first rate-limiting step in de novo lipogenesis, has attracted wide attention as a therapeutic target for non-alcoholic steatohepatitis (NASH). Typically, ACC1, an isoform of ACC, has been reported by Wang et al 12 as an independent prognostic indicator for HCC patients, and the ACC1-driven de novo FA synthesis promotes HCC cell survival, especially under the metabolic stress conditions, like glucose limitation or antiangiogenetic treatment. In addition, recent research has demonstrated that PPARα-SCD1 axis plays an important role in maintenance of the enriching cancer stem cells (CSC) properties of HCC sphere cells by promoting nuclear accumulation of β-catenin, 13 thereby producing novel views for the role of lipogenic enzymes in HCC. Inhibition of SCD1 interferes with sphere formation, down-regulated expression of CSC-related markers, and reduces β-catenin nuclear accumulation.
Although reduced FAO has been reported in many cases, some HCCs display a distinctly different metabolic phenotype characterized by a high β-oxidation rate. 8 Enhanced FAO, reduced glycolysis accompanied by the up-regulated expression of PPARα and CPT2 are observed in the β-catenin-activated HCCs derived from mice and humans (Figure 2), suggesting that such tumours rely mainly on FAO to provide energy. 8,9,14 Typically, the β-catenin-activated HCCs carry an activating mutation in the CTNNB1, a gene that encodes β-catenin in the Wnt pathway, whose mutation is not uncommon (19.5%) in human HCC. [15][16][17] Inhibiting FAO by genetic and pharmacological approaches blocks the HCC development, which suggests that inhibiting FAO is a suitable therapeutic approach for the β-catenin-mutated HCC. 14 In addition, Iwamoto et al recently reported that HCC cells would rather utilize FAO for their survival under the hypoxic conditions induced by the antiangiogenic drugs. Their results further showed that the oxygen and nutrient depletion induced by antiangiogenic drugs changed the glucose-dependent metabolism to the lipid-dependent metabolism through enhancing free FA uptake and the subsequent FAO, thus stimulating cancer cell proliferation. This phenomenon was mediated by the hypoxia-induced increased phosphorylation of activated protein kinase (AMPK), which increased FAO via increasing the CPT-1 activity by inducing the inhibitory phosphorylation of ACC2. 18 Recently, Bidkhori et al 19 had identified three HCC subtypes named iHCC1, iHCC2, and iHCC3 and pointed out that tumours in iHCC1 had the highest FAO fluxes, whereas 75% iHCC2 tumours carried mutations in CTNNB1, with up-regulated expression of β-catenin target genes (like glutamine synthetase GLUL and glutamate transporter SLC1A2). Finally, iHCC3 tumours were associated with the highest fluxes in FA biosynthesis and a strong Warburg effect.
Noteworthily, iHCC1 is the tumour group with the highest survival rate, which displays a high inflammation response compared with that of iHCC2 and is potentially associated with type 2 diabetes and obesity. iHCC3 tumours lead to the lowest survival rate and multiple malignant tumour features, including hypoxic behaviour and epithelial-to-mesenchymal transition (EMT). Therefore, it is speculated in this study that HCC patients with elevated FAO levels may have better prognosis, which is associated with higher inflammatory and immune responses. In addition, HCC with β-catenin mutation may be affected by lipotoxicity in a lipid-rich environment. On the other hand, the expression of phosphorylated AMPK is negatively correlated with the Ki-67 level (a cell proliferation marker), tumour grade and tumour size in HCC, as mentioned above, thus indicating that the high FAO level mediated by AMPK may be considered as a favourable factor. 20 In summary, these observations uncover the distinct differences in lipid metabolism within HCC that stem from the high inter-tumour heterogeneity, which are associated with patient survival.  Table 1.

| Uptake and transport of exogenous FAs in HCC
Although most cancer cells exhibit a metabolic shift towards lipogenesis and synthesize nearly all esterified FAs de novo, some tumours tend to "acquire" free FAs directly from the external environment. 35 External FAs are brought into the cell through a transport mechanism involving specialized enzymes and proteins such as FA translocase (FAT or CD36), fatty acid transport proteins FATP2 and FATP5, and members of FABP family (FABP1, FABP4 and FABP5), which are involved in FA uptake and transport in hepatic tissues as well as in HCC. 36,37 Increased expression of CD36 leads to higher FA uptake in HCC, which is closely related to induction of the mesenchymal transition (EMT). 36 The EMT may also be promoted when the enhanced FA levels of HCC patients up-regulate inflammation-related oncogenic transcriptional factors (NF-κB, AP-1, STAT3 and HIF-1α), which activate Wnt and TGF-β signalling pathways. 36,38,39 Given that depletion of FASN has been reported to significantly suppress HCC development, 40 there is also a great demand for novel therapeutic targets involved in lipid uptake and transport. In mouse hepatocytes, adenovirus-mediated knockdown of FATP2 or genetic deletion of FATP5 has been reported to significantly decrease the rates of fatty acid uptake, 41,42 which might have the potential to be new targets with regard to HCC. Taken together, more researches are needed to study more detailed mechanisms and verify the effect of deletion.

| Liver dysfunctions linked to HCC
Liver cancer displays a high incidence among people with chronic non-infectious liver diseases. Specifically, a non-esterified FAsrich condition may be a characteristic environment of obesity-and F I G U R E 2 Lipid metabolic reprogramming in β-catenin-activated HCC. Fatty acid β-oxidation (FAO) is activated to fuel HCC non-alcoholic steatohepatitis (NASH)-driven HCC ( Figure 3). 43,44 Nonetheless, it remains to be further explored about how HCC cells survive and grow in such an environment. Research shows that in human steatohepatitic HCC (SH-HCC), the expression of carnitine palmitoyltransferase 2 (CPT2), which converts acylcarnitine back to acyl-CoA, is down-regulated; subsequently, the marked accumulation of acylcarnitine species is detected, suggesting that the serum acylcarnitine levels may serve as a biomarker of HCC. 45 More importantly, CPT2 down-regulation suppresses the FAO pathway and enables HCC cells to escape from lipotoxicity to adapt to a lipid-rich environment, which is achieved through inhibiting the Src-mediated c-Jun NH2-terminal kinase (JNK) activation. 46 Furthermore, oleoyl carnitine (AC18:1), the long-chain acylcarnitine that accumulates through FAO suppression induced by CPT2 down-regulation, enhances hepatocarcinogenesis through the signal transducer and activator of transcription 3 (STAT3)-mediated acquisition of stem cell properties. 9,46 On the other hand, peroxisome proliferator-activated receptors (PPARs) are reported to be involved in regulating mitochondrial metabolism in liver within the disease context from NASH to HCC. The altered PPARs expression mainly induces mitochondrial metabolic dysfunctions, which can suppress FA oxidation, accumulate reactive oxygen species (ROS), and promote of lipogenesis. 47 The above evidence demonstrates that concomitant liver diseases may affect lipid metabolism and further promote tumour progression.  In terms of HCV-related HCC, the HCV core protein has been reported to play a vital part in enhancing the transcriptional activ-

| LIPID ME TABOLIC REPROG R AMMING OF IMMUNO C Y TE S IN H CC
The role of lipid metabolism in regulating immune cells has recently aroused general concerns. Evidence collected in several types of solid tumours indicated the importance of tumour immunometabolic reprogramming and suggested a novel and crucial area for future research of liver cancer. 48 The complicated crosstalk between metabolically reprogrammed immune cells and liver cancer cells has been suggested, but the molecular mechanisms need further exploration ( Figure 4).

| Tumour-associated macrophages
Macrophages are the versatile innate immunocytes, which con- which peaked with the increases in mitochondrial biogenesis and epigenetic reprogramming towards FAO. 71 Besides, triacylglycerol (TAG) uptake was also found to be essential for FAO in M2 macrophages, which was orchestrated by PPAR and liver-X-receptor (LXR). 72,73 In addition, apparently at odds with the increased FAO utilization, some TAMs accumulate intracellular lipids, which support not only their metabolic fitness, but also their immunomodulatory functions. Moreover, it has also been demonstrated that lipid loading of macrophages is associated with increased tumoricidal and

| T cells in TME
Recent studies have established that metabolic restrains, such as glucose restriction, impair the activities of effector T cells in TME. 69,83 In the same context, the remarkable expansion of acti- FAs content plays a crucial part in the levels of FAO and oxidative phosphorylation in cells, among which the former is a keyway for immunocytes to produce ATP. 87,88 Lipids are known to be the essential materials for cells, and their depletion in CD8 + T cells dramatically inhibits cell proliferation and signal transduction, which partly explains the lower number of CD8 + T cells in HCC than in adjacent tissues.
In terms of the related mechanism, the above-mentioned abnormal

| Other immune cells
Apart from the above-mentioned immunocytes, abnormal lipid metabolism is also reflected in the myeloid-derived suppressor cells  resulted in the susceptibility to sorafenib-induced hand-foot skin reaction (HFSR), which had revealed the novel candidate biomarkers.

| PER S PEC TIVE S OF H CC THER APY FROM LIPID ME TABOLIC REPROG R AMMING
In summary, abnormal lipid can potentially serve as a biomarker for diagnosing HCC.
As mentioned above, some HCC cells and immune cells recruited from TME meet their demands for energy and building materials by reductase (HMGCR)-driven cholesterol production is highly detrimental for HCC cell growth in culture. 119 In addition, some studies report that the increased SCD activity promotes hepatocarcinogenesis through accumulating monounsaturated fatty acids (MUFAs) and activating the unfolded protein response via the ER stress related to sorafenib resistance in HCC. In addition, the researchers believe that SCD inhibition may reshift the balance of FA composition towards saturation, which exert a synergistic effect on HCC with sorafenib. 120 Besides, inhibition of FAO by etomoxir or other methods (such as targeted delivery of CPT1A siRNA/shRNA) may limit the immunosuppressive function of M2 macrophages, which may be developed as an effective therapeutic strategy. 48 Some immunotherapies related to lipid metabolism for cancers were summarized in Table 2. Nonetheless, in consideration of the genotypic and tumour-biological diversities of HCC patients, as well as the complex lipid metabolism, therapeutic strategies targeting the FArelated pathways against HCC still lag behind.

| CON CLUS IONS
Our understandings towards the lipid metabolic changes in HCC development have been improved remarkably over the past years.
Nevertheless, the impacts of dysregulated FAs metabolism on HCC cells and the TME remains incompletely known So far here, and faeces, and reduced plaque size and lipid content in mice. [121][122][123] More attention should be paid to miRNA therapeutics for HCC with aberrant lipid metabolism. Afterwards, as we mentioned above, studies on exogenous lipid uptake and transport should also be further carried out to obtain new therapeutic targets in an HCC setting. In addition, the mTOR pathway may be a suitable target to regulate immune cells by manipulating cellular lipid metabolism. Inhibition of mTOR by rapamycin can block the development of macrophages and CD4 + T cells in several scenarios, including liver cancer. 124,125 Future studies can make efforts to combine immunity and lipid metabolism, so as to develop novel therapeutic methods that can benefit patients with HCC.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

AUTH O R S ' CO NTR I B UTI O N S
XS and JL created the idea for the review. BH performed the selection of literature, drafted the manuscript, and prepared the figures.
XY and JL revised the manuscript. All authors read and approved the final manuscript.

CO N S E NT FO R PU B LI C ATI O N
All authors agree to submit for consideration for publication in the journal.
TA B L E 2 Immunotherapy related to lipid metabolism for cancers