**4. Discussion**

The analysis of the mutational landscape revealed that TP53, KRAS (Kirsten rat sarcoma viral oncogene), IDH1 (Isocitrate dehydrogenase 1), and PTEN (Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase) are the most frequently mutated genes in intrahepatic CCCs [26]. Numerous studies have shown that p53 is involved in the regulation of many reactions in energy metabolism. p53 can be regarded as a master regulator of energy metabolism since it influences glycolysis, gluconeogenesis, the pentose-phosphate pathway, mitochondrial OXPHOS, and glutamine metabolism [27,28]. Mutations in the oncogene KRAS drive metabolic reprogramming through enhanced glucose uptake and regulation of glutamine metabolism [29]. Additionally, PTEN was shown to regulate several aspects of energy metabolism [30]. IDH1 catalyzes the reversible oxidative decarboxylation of isocitrate to yield α-ketoglutarate ( α-KG). Under hypoxic conditions, IDH1 catalyzes the reverse reaction of α-KG to isocitrate, which contributes to citrate production via glutaminolysis [31,32]. In the present study, the levels of SDHA, UQCRC2, MT-CO1 and ATP5F1A were significantly reduced in the tumor periphery compared to the control tissue. Thus, genetic alterations of proteins influencing energy metabolism are clearly central to the pathogenesis of CCC.

Large areas of NDUFS4 negative cells were found in tumors and control tissue, suggesting that this is an early event in tumorigenesis. No patient had large negative regions of MT-CO1 or ATP5F1A in normal tissue. In addition, SDHA and UQCRC2 were a ffected in only 11% and 10% of the cases, respectively. Mitochondrial DNA was reported to be significantly mutated in CCCs [26,33]. This can partially explain the observed OXPHOS defects in our sample cohort. Moreover, the heteroplasmy of mtDNA mutations might explain the heterogeneity of the OXPHOS subunit expression [34]. According to the COSMIC database, potentially pathogenic mutations in nuclear-encoded OXPHOS subunits are very rare events in CCCs. Therefore, it would be interesting to analyze the mutational landscape (TP53, KRAS, IDH1, PTEN) in relation to the expression of OXPHOS subunits. However, it was not possible to perform a detailed genetic analysis because this was a retrospective study using formalin-fixed para ffin-embedded (FFPE) tissue.

As we found di fferences in the expression levels between the tumor center and periphery, we hypothesize that within CCCs, several modes of energy generation coexist. Tumor cells at the margin might be more dependent on glucose than tumor cells at the center, as indicated by the lower levels of OXPHOS subunits in the latter. This could be attributable to the fact that newly generated tumor cells at the growth front might not be su fficiently supplied with oxygen, because the generation of new blood vessels requires time. However, this hypothesis is controversial because necrosis is often seen in the tumor center, which is at least partially attributed (in the literature) to low oxygen supply.

Mitochondrial mass was increased in the tumor center compared to adjacent normal tissue, as indicated by VDAC1 staining. A significant inverse correlation found between the percentage of VDAC1-positive cells and MT-CO1-negative cells suggests there may be compensatory upregulation of mitochondrial mass. This phenomenon is well described in individuals with mitochondrial disorders, who frequently have increased mitochondrial content, as indicated by citrate synthase activity, VDAC1 levels, and mtDNA copy number [35]. In addition, oncocytic tumors, which are characterized by complex I defects caused by pathogenic mutations in mtDNA-encoded subunits, show a very pronounced increase in mitochondrial mass [19,36]. In addition, mtDNA content and mass increase in the tumorigenic progression of normal endometrial tissue to hyperplastic tissue to cancerous tissue [37]. MtDNA mutations and deletions have been reported in endometrial tumors [38].

Another possibility is that tumor cells at the periphery might express di fferent sets of proteins compared to tumor cells in the center. The intercellular or cell–cell lactate shuttle hypothesis proposes that lactate is generated and exported from one cell and taken up and utilized by another cell. This mechanism has been described for neurons and astrocytes [39].

A significant inverse correlation was found between VDAC1 expression and UICC tumor stage. This agrees with findings in the literature, and may be explained as follows: aggressive tumors that are highly dependent on glucose should potentially exhibit low mitochondrial mass. In support, we found that VDAC1 levels were lower in cases with lymph node involvement. The same trend to lower VDAC1 levels was also present if metastasis occurred. Significantly lower survival was observed for low/moderate VDAC1 expressors compared to high expressors. That is, lower mitochondrial mass was associated with shorter survival.

The clear association between energy metabolism and CCC development has important therapeutic implications. CCCs might be susceptible to metabolic therapies such as the ketogenic (high-fat, low-carbohydrate) diet, which was recently shown to significantly inhibit tumor growth in numerous xenograft models and patients [40–43].

**Supplementary Materials:** The supplementary materials are available online at http://www.mdpi.com/2073-4409/ 8/6/539/s1.

**Author Contributions:** R.G.F. stained samples, analyzed data and wrote the first draft of the manuscript. D.N. and R.K. analyzed the IHC staining, prepared the tissue array and critically reviewed the manuscript. T.K. analyzed the IHC staining and provided material. B.K. and J.A.M. supervised the project. All authors discussed the results and commented on the manuscript.

**Funding:** This research was supported by grants from the "Vereinigung zur Förderung der pädiatrischen Forschung und Fortbildung Salzburg" and the Austrian Research Promotion Agency (822782/THERAPEP).

**Conflicts of Interest:** The authors declare no conflict of interest.
