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Correnti et al. Hepatoma Res 2018;4:69 I http://dx.doi.org/10.20517/2394-5079.2018.96 Page 7 of 15

explanation for the different metastatic activity observed between CSCs and other tumor cells might be
[122]
the EMT status of CSCs, which enables them to have a prominent role in the metastasis and invasion .
Malignant cells undergo molecular changes typical of EMT, which represents a key stage of the metastatic
multistep process, and eventually undergo a mesenchymal-to-epithelial transition (MET) to generate
secondary tumors in target organs. Hence, CSCs mediate tumor metastasis by maintaining plasticity
to transition between epithelial or mesenchymal states, and the EMT process represents the potential
link between CSCs and circulating metastasis-initiating cells [121,133] . For example, in the CCA cell line
TFK-1, TGF-β1 is able to induce not only EMT, but also CSC generation with a consequent decreased
sensitivity to the chemotherapeutic agent 5-FU. Furthermore, the EMT-related overexpression of hepatic
transmembrane 4L six family member 5 (TM4SF5) has a potential role in generating HCC cCSCs with
metastatic properties through interaction with CD44 [121,134] . In addition, HCC CSCs isolated by sphere
assay are associated with an enhanced expression of the variant isoforms of CD44, which are related to
CSC chemo-resistance, as well as with an increased frequency of intrahepatic metastasis when injected
null
null
in the spleen of NOD-Rag1 IL2rγ double mutant mice (NRG mice). Also in this case, enhancement
of the EMT correlates to the metastatic potential and CSC state [135] . Another study has revealed that
CD44 is associated with a mesenchymal phenotype in HCC cell lines, and knockdown of CD44 reverses
[136]
EMT and inhibits lung metastasis of HCC cells in a murine model . Another gene expression analysis
of microarray data from 238 HCC cases has revealed an enriched EMT signature in CD90+ stem-like
[137]
cells . Finally, a recent study has found that CD44 protein levels are enhanced after TGF-β1 treatment
and that interaction between CD44 and TGF-β1 induces EMT and CSC phenotypes through β-catenin
[138]
signaling in HCC . All these findings strengthen the hypothesis of an existing link between EMT and
CSC cellular states in relation with the metastatic process.

Metabolic reprogramming
Starting from the pioneering work of Otto Warburg, several observations have indicated that tumor genetic
alterations imply also cell metabolism reorganization [139,140] . In particular, it has been shown that tumor
cells produce ATP via glycolysis and accumulate extracellular lactate even under normoxic conditions [140,141] ,
[140]
and often present a limited or absent mitochondrial oxidative phosphorylation (OXPHOS) . Although
metabolic reprogramming is currently considered a hallmark of cancer, no consensus has been reached
on the metabolic features of CSCs. which are very plastic and capable of either reside in a dormant state,
or rapidly proliferate to replenish the tumor mass. A number of studies suggest that CSCs more strongly
favor the glycolytic pathway compared to bulk tumor cells, while other studies report that mitochondrial
[141]
oxidative metabolism is the prevalent source of energy for CSC (reviewed in ) [Figure 2]. However, even
if investigation of PLC metabolism is still at its very beginning in comparison with other tumor systems,
recent evidence has revealed the importance of the metabolic rearrangement in PLC CSCs. CD44+ CCA
CSCs adapt their redox status regulation according to their needs and contribute to reactive oxygen
species (ROS) defense promoting glutathione synthesis by way of xCT (a cysteine-glutamate transporter),
[142]
resulting in evasion of cell death . Moreover, CD133+ HCC CSCs are characterized by high glycolytic
metabolism with concomitant overexpression of glycolytic genes and enhanced extracellular acidification
rate, demonstrating that CD133+ cells are more glycolytic compared to CD133- cells. Further, CD133+
[143]
cells stemness features are significantly reduced when glycolysis is inhibited . Extensive trascriptome
and metabolome analysis of CD133+ HCC cells revealed the key role of MYC in the regulation of
[144]
glycolytic metabolism in HCC CSCs .

There is also an increasing interest in lipid metabolism and specifically in alterations in lipid and
cholesterol-associated pathways. It is well known that proliferating tumor cells require lipids and
cholesterol, and they may increase the uptake of exogenous lipids and lipoproteins or hyper-activate
metabolic pathways deputed to produce lipids and cholesterol. When specifically looking at the stem cell
compartment, it has been demonstrated that stem-like cells rely on fatty acid oxidation (FAO) for the
[145]
generation of ATP and NADH [Figure 2]. Metabolism analysis has revealed that NAD+ concentrations