In the current study, combined the transcriptomics and proteomics data from two databases, namely TCGA and CNHPP, with the protein abundance and metabolomic signature from our own datasets of HBV-associated HCC corhort, we determined the metabolic reprogramming taking place in HBV-associated HCC, such as activated sphingolipid metabolism which correlated with poor prognosis of HCC, and the perturbation in linoleic acid metabolism and primary bile acid metabolism signaling pathway in a virus load dependent manner. The tissue metabolome partially verified the results from serum metabolome, and identified the significant elevated unsaturated fatty acids. Additionally, the protein array probed the major energy metabolism signaling pathway showed a significant changes along the PI3/AKT/mTOR signaling cascade (as shown in Fig. 7).
Firstly, HBV resulted in a distinct metabolic signature of HCC to gain survival advantage. And HBV positive and negtive HCC should be treated with different interventions. With the development in sequencing-based studies, the genetic landscape of HBV-related HCC has been uncovered comprehensively . However, the effect of HBV on the cancer progression remains unclear. By using the transcriptomic data from TCGA, we obtained 738 DEGs compared HBV-positive HCC patients with HBV-negative HCC patients. Upregulated genes such as LGALS14, BPIFA1, MDBT1, HHATL have been reported for their correlation with the overall survival of HCC patients [20–23], while the decreased PCSK1, SST, CLCA1, ZNF385D-AS2, SOX1 predicted a poor overall survival as previous publications reported [24–26]. Previous publication claimed that the metabolic alterations in all primary liver cancer might be metabolically similar, involved metabolic pathways such as glucose metabolism, nucleotide metabolism, amino acid metabolism and lipid metabolism . Our pathway annotation based on metabolic profile (HBV-positive HCC vs HBV-negative HCC) showed that the metabolic alterations mainly attributed to cholesterol metabolic process (-log 10(P)=8.3), lipid biosynthetic process (-log 10(P)=7). By observing the in vitro metabolites profile, the influence of HBV on tumorgenesis was attributed to induction of central carbon metabolism, including the hexosamine biosynthesis pathway, phosphatidylcholine synthesis, activation of de novo lipogenesis .
Primary bile acid metabolism, steroid hermone and the sphingolipid metabolism were influenced by HBV. Previous studies have reported not only the elevation of bile acid in HCC patients compared with cirrhosis counterparts, also the induction in HBV-infected mice, showed positive correlation of bile acid with HCC, and the HBV-infected animal model [29, 30]. Pathway annotation of metabolites profile in our studies also suggested that the primary bile acid biosynthesis was of higher activity compared to the HBV-negative group. Moreover, in HBV-positive subgroup possessing higher virus load, the metabolites in the primary bile acid biosynthesis pathway were increased compared to HBV-positive patients who possess lower virus load. Metabolic profile from HBV-positive HCC biopsies also showed the abundance of metabolites in bile acid biosynthesis process, including cholic acid, chenodeoxycholic acid, and related intermittent metabolites (T vs. NT). Moreover, the transportation of bile acids within hepatocyte is highly dependent on Na+-taurocholate Co-transporting Polypeptide (NTCP), which was initially known as HBV entry receptor (Molecular regulation of the hepatic bile acid uptake transporter and HBV entry receptor NTCP). All together, these results pointed that bile acid metabolism could be a promising target for HBV-positive HCC patients.
Steroid hormones such as androgens, estrogens has been reported play a pivotal role in the progression of HBV infection and the development of HBV-related HCC, mainly via inducing epigenetic regulation such as the regulation of mRNA levels by microRNAs, DNA methylation . Moreover, liver could regulated the synthesis and bioactivity of steroid hormones, which reversely contribute to the proper liver function . For example, testosterone can influence the hepatic lipid deposition, via regulating lipogenic enzyme .
Sphingolipids have been reported could help form cellular membrane, contribute to its flexibity and membrane trafficking . The changes of serum sphingolipids in HBV infection and cancer progression was controversial. Some claimed that the increase of sphingolipid in HBV infection and cancer progression, however others reported that most of the sphingolipids were decreased in HBV-positive HCC patients compared with HBV-associated cirrhosis [35, 36]. In our study, not only the upregulation of sphingolipids was observed in HBV-positive HCC compared with HBV-negative HCC, also the activation of spingolipid metabolism pathway was correlated with a poor prognosis shown by ssGSEA analysis. Moreover, tissue metabolomics showed that the sphingomyelin was higher in biopsies from HBV infected patients (Fig. 6a), so as the lipid profile showed the perturbation of ceramides (Fig. 6d). The upregulation of sphingolipids might contribute to the HBV-related HCC progression. Elevated PDK1 which has been reported corrleated with radiation-resistant of HCC , thus the activation of PDK1 in HBV-positive HCC patients probably indicated a poor prognosis.
HBV-positive HCC patients showed an altered lipid composition, and accumulation of unsaturated fatty acids. The up-regulated hexosamine and phosphatidylcholine from a cell model (hepG2.2.15) was observed as the HBV stimulated metabolic changes. However, how liver injury and HCC may be accelerated in patients with HBV infection, and which metabolic pathways are being identified as potential targets to counteract or treat cancer remains unclear. Our results indicated that HBV-positive HCC patients bearing a unique characteristic particularly in lipids categories, such as fatty acyl, glycerophospholipids, steorl lipids, spingolipids. The abundance of lipid metabolites belongs to primary bile acid, sphingolipids has been discussed above. Aside from the bile acids and sphingolipids, the glycerophospholipids, such as lysophosphocholine 18:2 was higher in HBV-positive HCC biopsy compared with either the non-cancerous tissue from same patients, or the HBV-negative HCC biopsy. Additionally, phosphorylethanolamine 18 showed decrease in HBV-positive NT compared with HBV-negative NT. The accumulation of unsaturated fatty acids has been reported correlated with tumorogenesis, also could interact with immune regulators, thus predicted a poor prognosis in HCC [38, 39]. As our data showed that unsaturated fatty acids, such as linoleic acid, vaccenic acid showed a reducing trend in biopsy of HBV-positive patients compared with the adjacent noncancerous tissue. Additionally, 9z,12z-linoleic acid, α-linolenic acid, trans-vaccenic acid, oleic acid, 9-trans-palmitelaidic acid, nervonic acid were increased in HBV-positive hepatic compared with that of HBV-negative, indicated that the unsaturated fatty acid in tumor microenvironment is essential for cancer progression .
PI3K/Akt/mTOR resulted lipogenesis partially reponsible for metabolic alteration in HCC.As one of the most frequently dysregulated signalling pathway, PI3K/AKT signaling, could stimulate growth, proliferation and survival. The intricate regulation of lipid homoestasis of PI3K/AKT has been reported, not only limited to intracellular process, such as membrane synthesis, but also remodeling the tumor microenvironment by paracine mechanism . Upon PI3Kα recruited to plasma membrane, sequentially phosphorylated phosphatidylinositol (4,5)-bisphosphate and phosphatidylinositol (3, 4,5)-bisphosphate, and subsequent activation of AKT, which ultimately promote the stability of SREBP1c and lipogenesis. As the antibody array showed a hyperactivated PI3K/AKT signaling, particulary the upregulation of 4E-BP1 which involved in the mTORC1 dependent glycolytic pathway, consistent with previous publication about Hepatitis B virus (HBV) pre-S2 mutant transgenic HCC mice model (4E-BP1, a multifactor regulated multifunctional protein). Additionally, the alternative regulation of mTOR, was the GSK3β promoted 4E-BP1 phosphorylation . The induction of Raf1 by HBV has been reported previously, the activation of Raf/MEK/ERK signaling play a key role in HCC tumorgenesis and development [43, 44]. Our results showed upregulation of Raf1 in biopsy of HBV-positive HCC further indicated Raf1 as a promisng target for HBV-HCC patients.
By multi-omics platforms, the metabolic phenotype of HCC, has been extensively investigated [45, 46]. Recently, by linking the metabolites and protein interaction two HCC subtypes were identified, the poor prognostic subtype showed more fatty acid accumulation, however there is no significant difference of HBV infection history among the two groups . These development on omics based approaches have improved our recognition in cancer metabolism, but existing tecnnique can not quantitatively and qualitatively reveal the whole landscape of the metabolism until now. Further experiments should be carried out in the future to validate the detailed metabolic reprogramming and the regulation mechanism.