Role of specific metabolic pathways in clear cell Renal Cell Carcinoma (ccRCC)
Clear cell Renal Cell Carcinoma (ccRCC) is the most frequent subtype of renal cancer, and is histologically characterized by cells with clear cytoplasm due to the marked presence of lipid and glycogen. In about 90% of its sporadic forms the bi-allelic inactivation of VHL prevents degradation of hypoxia-inducible factor-1a and 2a (HIF1a, HIF2a) with constitutive activation of their function . HIF1 and HIF2, through the regulation of different and specific hypoxia-inducible genes, have an important role in the development of various metabolic alterations  responsible also of the “clear” cytoplasm that characterizes the cells of this tumor. The activation of glycolysis and lactate production even in the presence of oxygen (Warburg effect), the alteration of mitochondrial oxidative metabolism, and the switch of glutamine metabolism that supply Krebs cycle to support lipogenesis, are hallmarks of the metabolic reprogramming in ccRCC  and also potential therapeutic targets of this poorly treatable disease. Interestingly, PPAR pathways, involved in fatty acid metabolism  and in adipocyte differentiation characterized by lipid droplet accumulation, have been recently described as target to inhibit ccRCC cell line growth . Moreover, PPAR-dependent adipocyte differentiation, seems to be negatively regulated by Annexin A3 protein  that we have previously evidenced as downregulated in RCCcc cells .
Based on these data, our aim was to investigate, by cytological, molecular, and functional approaches whether: 1) lipid and glycogen storages were differently modulated on the basis of histopathological grading of ccRCC; 2) the viability of ccRCC cells obtained from tumor tissues with different Fuhrman grade was differently affected by specific metabolic pathway inhibition; 3) Annexin A3 was involved in lipid storage of ccRCC cells.
Methods and results
Primary cell cultures, established from ccRCC and normal cortex tissue samples, were characterized by FACS and immunofluorescence analysis. Lipid and glycogen storages into ccRCC primary cultures and in corresponding tissues of origin was evaluated by Oil Red “O” and PAS staining, respectively. We observed that, like in corresponding tumors, the lipid storages were more abundant in lower grade (G2) than in higher grade (G3-G4) ccRCC primary cultures, in which the lipid droplets were also smaller. On the contrary, the glycogen storages were more abundant in higher grade (G3-G4) ccRCC cultures and tissues. Based on these data, we analyzed by MTT assay the viability of primary cell cultures obtained from different grade ccRCCs after 72 h of treatment with specific inhibitors of lipid (etomoxir) or glucose (2DG) metabolism and after culture in glutamine depleted media. We evidenced that the viability of ccRCC cultures obtained from low grade tumors (G2) were affected by treatments that interfered with lipid metabolism (etomoxir and glutamine depletion). Instead, ccRCC culture viability from higher grade tumors (G3) was affected by treatments that interfered with glucose metabolism (2DG). Also transcriptomic and miRNA analysis of our ccRCC primary cell cultures showed, respect to normal cortex primary cultures, a differential expression of genes involved in lipid and glucose metabolism consistent with the cytological phenotype observed. One example is provided by PLIN2 gene that code for a protein that is expressed on lipid droplets and is a marker of lipid accumulation. In fact, transcriptomic analysis and PCR validation revealed that PLIN2 was upregulated in our ccRCC cells respect to normal cortex cells (Fig.1). Moreover, we observed in ccRCC cell lines that a high expression level of Annexin A3 protein, evaluated by western blot, correlated with a more abundant lipid storage, evaluated by Oil Red staining quantification. In addition, Annexin A3 gene silencing by siRNA induced in these ccRCC cell lines an increase of lipid storage with a decrease of cell viability. These data seem to evidence a possible involvement of Annexin A3 in modulation of lipid metabolism and storage in ccRCC cells.
In conclusion, these data evidence a different metabolic storage in ccRCC cells and a different impact of glucose and lipid metabolism on their cell viability in relation to histopathological grading of corresponding tumor tissues. These results, confirmed in a wider series and read over for HIF1a and HIF2a expression pattern, might be useful to develop an approach to “personalized medicine” for ccRCC. Specific metabolic pathways, highly represented in specific RCC grades, could be targeted with therapies that ablate those pathways. Moreover, the putative involvement of Annexin A3 protein in modulation of lipid storage in ccRCC cells may help to shed light on the complex molecular mechanisms involved in metabolic reprogramming of ccRCC.
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