Impact of targeting MYC in metabolic reprogramming and differentiation of cancer

Sammanfattning: Alterations in several metabolic pathways due to increased energy and biomass demand, as consequence of the uncontrolled proliferation of cancer cells, is known as metabolic reprogramming. Mutations in tumor suppressor genes and oncogenes that initiate cancer development, are responsible directly and indirectly of the changes in major cellular energy production processes, including glycolysis, glutaminolysis, and lipid metabolism. Neuroblastoma (NB) is a solid tumor that develops extracranially in the sympathetic nervous system, and the most diagnosed cancer during the first year of life. Among several genetic alterations, MYCN-amplification occurs in approximately 25% of all cases, associated with poor survival rate. Although the MYCN protein plays a crucial role in NB progression, no inhibitors have been approved for clinical use, as targeting MYCN has proven to be challenging. Thus, other indirect strategies, such as targeting downstream processes controlled by MYCN including metabolism and differentiation, represent alternatives to overcome the drawbacks of directly targeting this oncoprotein. In clear cell renal cell carcinoma (ccRCC), loss of the von Hippel-Lindau (VHL) gene provokes constitutive activation of hypoxia inducible factors (HIFs). Due to disruptions in a myriad of metabolic pathways, and in part, as consequence of the continuous stabilization of HIFs, ccRCC is considered a metabolic disease. Moreover, although MYC amplification is found only in 5-10 % of the cases, increased MYC signaling has been associated with development of aggressive forms of ccRCC. In paper I we investigated the metabolic changes induced by MYCN amplification in NB. By combining proteomics, transcriptome analysis and functional metabolic assays, we demonstrated that MYCN induced changes in several metabolic enzymes, increasing glycolysis and oxidative phosphorylation. We also found that fatty acids were the preferred mitochondrial fuel for energy production in MYCN-amplified cells. Moreover, data from tracing experiments with 13C-labeled glucose or glutamine indicated that MYCN-amplified NB cells synthetized glutamine de novo. Furthermore, targeting fatty acid oxidation resulted in reduction of viability in NB cells with MYCN-amplification in vitro and in reduction of tumor burden in vivo. Since we found that fatty acid oxidation was relevant for MYCN-amplified NB, we further studied the effects of inhibiting de novo fatty acids synthesis in paper II. Using five different inhibitors targeting two consecutive enzymes in the process, we described that inhibition of the synthesis of fatty acids resulted in striking neuronal differentiation associated with activation of ERK signaling, and reduction of MYCN and MYC levels. Moreover, lipid composition as well as mitochondrial function and morphology of NB cells was altered. In addition, fatty acid synthesis inhibition led to reduced tumor formation and increased differentiation markers in several NB xenograft models. Together, the results in paper I and II suggested that targeting lipid metabolism could be a potential therapeutic approach for NB patients. In paper III we further analyzed the potential differentiation of NB cells induced by activation of nuclear hormone receptors (NHRs). Our data showed that the simultaneous activation of glucocorticoid receptor (GR), estrogen receptor α (ERα) and retinoic acid receptor α (RARα) potentiated neurite outgrowth, induced changes in the glycolytic and mitochondrial functions, accompanied with lipid droplet accumulation, and reduced proliferation in vitro as well as tumor burden in vivo. In addition, single cell nuclei analysis revealed a sequential expression of the three NHRs during adrenal gland development. Notably, in silico analysis of patient cohorts demonstrated that high expression of these NHRs were correlated with better overall survival. Thus, combination therapy with the concurrent activation of GR, ERα and RARα represents a promising strategy to induce differentiation in NB patients. Paper IV describes the mechanism behind lipid droplet (LD) accumulation induced by MYC inhibition during hypoxia in clear cell renal cell carcinoma (ccRCC). We found that HIF expression together with MYC inhibition resulted in LD deposition. Our results showed that due to HIF stabilization, glutamine-derived carbons were directed for synthesis of fatty acids, further accumulating in LDs. Importantly, we identified that the hypoxia inducible lipid droplet associated (HILPDA) gene, was overexpressed upon HIF induction and MYC inhibition, controlling LD formation in ccRCC cells. Hence, our study characterizes the molecular mechanism of LD accumulation in relation to hypoxia and MYC signaling, providing new understanding of metabolic adaption in ccRCC. Altogether, the data compiled in this thesis describes the important role of the MYC family of proteins in differentiation and metabolism of NB and in the metabolic reprogramming of ccRCC providing new knowledge and potential targets for development of novel therapeutic approaches.