During decades, the research field of cancer metabolism was based on the Warburg effect, described almost one century ago. Lately, the key role of mitochondria in cancer development has been demonstrated. Many mitochondrial pathways including oxidative phosphorylation, fatty acid, glutamine, and one carbon metabolism are altered in tumors, due to mutations in oncogenes and tumor suppressor genes, as well as in metabolic enzymes. This results in metabolic reprogramming that sustains rapid cell proliferation and can lead to an increase in reactive oxygen species used by cancer cells to maintain pro-tumorigenic signaling pathways while avoiding cellular death. The knowledge acquired on the importance of mitochondrial cancer metabolism is now being translated into clinical practice. Detailed genomic, transcriptomic, and metabolomic analysis of tumors are necessary to develop more precise treatments. The successful use of drugs targeting metabolic mitochondrial enzymes has highlighted the potential for their use in precision medicine and many therapeutic candidates are in clinical trials. However, development of efficient personalized drugs has proved challenging and the combination with other strategies such as chemocytotoxic drugs, immunotherapy, and ketogenic or calorie restriction diets is likely necessary to boost their potential. In this review, we summarize the main mitochondrial features, metabolic pathways, and their alterations in different cancer types. We also present an overview of current inhibitors, highlight enzymes that are attractive targets, and discuss challenges with translation of these approaches into clinical practice. The role of mitochondria in cancer is indisputable and presents several attractive targets for both tailored and personalized cancer therapy.

The MAX network transcriptional repressor (MNT) is an MXD family transcription factor of the basic helix-loop-helix (bHLH) family. MNT dimerizes with another transcriptional regulator, MYC-associated factor X (MAX), and down-regulates genes by binding to E-boxes. MAX also dimerizes with MYC, an oncogenic bHLH transcription factor. Upon E-box binding, the MYC–MAX dimer activates gene expression. MNT also binds to the MAX dimerization protein MLX (MLX), and MNT–MLX and MNT–MAX dimers co-exist. However, all MNT functions have been attributed to MNT–MAX dimers, and no functions of the MNT–MLX dimer have been described. MNT's biological role has been linked to its function as a MYC oncogene modulator, but little is known about its regulation. We show here that MNT localizes to the nucleus of MAX-expressing cells and that MNT–MAX dimers bind and repress the MNT promoter, an effect that depends on one of the two E-boxes on this promoter. In MAX-deficient cells, MNT was overexpressed and redistributed to the cytoplasm. Interestingly, MNT was required for cell proliferation even in the absence of MAX. We show that in MAX-deficient cells, MNT binds to MLX, but also forms homodimers. RNA-sequencing experiments revealed that MNT regulates the expression of several genes even in the absence of MAX, with many of these genes being involved in cell cycle regulation and DNA repair. Of note, MNT–MNT homodimers regulated the transcription of some genes involved in cell proliferation. The tight regulation of MNT and its functionality even without MAX suggest a major role for MNT in cell proliferation.

MXD1 is a protein that interacts with MAX, to form a repressive transcription factor. MXD1-MAX binds E-boxes. MXD1-MAX antagonizes the transcriptional activity of the MYC oncoprotein in most models. It has been reported that MYC overexpression leads to augmented RNA synthesis and ribosome biogenesis, which is a relevant activity in MYC-mediated tumorigenesis. Here we describe that MXD1, but not MYC or MNT, localizes to the nucleolus in a wide array of cell lines derived from different tissues (carcinoma, leukemia) as well as in embryonic stem cells. MXD1 also localizes in the nucleolus of primary tissue cells as neurons and Sertoli cells. The nucleolar localization of MXD1 was confirmed by co-localization with UBF. Co-immunoprecipitation experiments showed that MXD1 interacted with UBF and proximity ligase assays revealed that this interaction takes place in the nucleolus. Furthermore, chromatin immunoprecipitation assays showed that MXD1 was bound in the transcribed rDNA chromatin, where it co-localizes with UBF, but also in the ribosomal intergenic regions. The MXD1 involvement in rRNA synthesis was also suggested by the nucleolar segregation upon rRNA synthesis inhibition by actinomycin D. Silencing of MXD1 with siRNAs resulted in increased synthesis of pre-rRNA while enforced MXD1 expression reduces it. The results suggest a new role for MXD1, which is the control of ribosome biogenesis. This new MXD1 function would be important to curb MYC activity in tumor cells.

MNT is a crucial modulator of MYC, controls several cellular functions, and is activated in most human cancers. It is the largest, most divergent, and most ubiquitously expressed protein of the MXD family. MNT was first described as a MYC antagonist and tumor suppressor. Indeed, 10% of human tumors present deletions of one MNT allele. However, some reports show that MNT functions in cooperation with MYC by maintaining cell proliferation, promoting tumor cell survival, and supporting MYC-driven tumorigenesis in cellular and animal models. Although MAX was originally considered MNT’s obligate partner, our recent findings demonstrate that MNT also works independently. MNT forms homodimers and interacts with proteins both outside and inside of the proximal MYC network. These complexes are involved in a wide array of cellular processes, from transcriptional repression via SIN3 to the modulation of metabolism through MLX as well as immunity and apoptosis via REL. In this review, we discuss the present knowledge of MNT with a special focus on its interactome, which sheds light on the complex and essential role of MNT in cell biology.

MNT, a transcription factor of the MXD family, is an important modulator of the oncoprotein MYC. Both MNT and MYC are basic-helix–loop–helix proteins that heterodimerize with MAX in a mutually exclusive manner, and bind to E-boxes within regulatory regions of their target genes. While MYC generally activates transcription, MNT represses it. However, the molecular interactions involving MNT as a transcriptional regulator beyond the binding to MAX remain unexplored. Here we demonstrate a novel MAX-independent protein interaction between MNT and REL, the oncogenic member of the NF-κB family. REL participates in important biological processes and it is altered in a variety of tumors. REL is a transcription factor that remains inactive in the cytoplasm in an inhibitory complex with IκB and translocates to the nucleus when the NF-κB pathway is activated. In the present manuscript, we show that MNT knockdown triggers REL translocation into the nucleus and thus the activation of the NF-κB pathway. Meanwhile, MNT overexpression results in the repression of IκBα, a bona fide REL target. Both MNT and REL bind to the IκBα gene on the first exon, suggesting its regulation as an MNT–REL complex. Altogether our data indicate that MNT acts as a repressor of the NF-κB pathway by two mechanisms: (1) retention of REL in the cytoplasm by MNT interaction, and (2) MNT-driven repression of REL-target genes through an MNT–REL complex. These results widen our knowledge about MNT biological roles and reveal a novel connection between the MYC/MXD and NF-κB pathways, two of the most prominent pathways in cancer.

Background Neuroblastoma (NB), a childhood tumor derived from the sympathetic nervous system, presents with heterogeneous clinical behavior. While some tumors regress spontaneously without medical intervention, others are resistant to therapy, associated with an aggressive phenotype. MYCN-amplification, frequently occurring in high-risk NB, is correlated with an undifferentiated phenotype and poor prognosis. Differentiation induction has been proposed as a therapeutic approach for high-risk NB. We have previously shown that MYCN maintains an undifferentiated state via regulation of the miR-17 ~ 92 microRNA cluster, repressing the nuclear hormone receptors (NHRs) estrogen receptor alpha (ERα) and the glucocorticoid receptor (GR). Methods Cell viability was determined by WST-1. Expression of differentiation markers was analyzed by Western blot, RT-qPCR, and immunofluorescence analysis. Metabolic phenotypes were studied using Agilent Extracellular Flux Analyzer, and accumulation of lipid droplets by Nile Red staining. Expression of angiogenesis, proliferation, and neuronal differentiation markers, and tumor sections were assessed by immunohistochemistry. Gene expression from NB patient as well as adrenal gland cohorts were analyzed using GraphPad Prism software (v.8) and GSEA (v4.0.3), while pseudo-time progression on post-natal adrenal gland cells from single-nuclei transcriptome data was computed using scVelo. Results Here, we show that simultaneous activation of GR and ERα potentiated induction of neuronal differentiation, reduced NB cell viability in vitro, and decreased tumor burden in vivo. This was accompanied by a metabolic reprogramming manifested by changes in the glycolytic and mitochondrial functions and in lipid droplet accumulation. Activation of the retinoic acid receptor alpha (RARα) with all-trans retinoic acid (ATRA) further enhanced the differentiated phenotype as well as the metabolic switch. Single-cell nuclei transcriptome analysis of human adrenal glands indicated a sequential expression of ERα, GR, and RARα during development from progenitor to differentiated chromaffin cells. Further, in silico analysis revealed that patients with higher combined expression of GR, ERα, and RARα mRNA levels had elevated expression of neuronal differentiation markers and a favorable outcome. Conclusion Together, our findings suggest that combination therapy involving activation of several NHRs could be a promising pharmacological approach for differentiation treatment of NB patients.

IntroductionMNT has been described as an antagonist and modulator of MYC, one of the most prevalent oncoproteins in human cancer. Both MYC and MNT are bHLH-LZ transcription factors that heterodimerize with MAX, bind to E-boxes within regulatory regions of target genes, and generally activate (MYC) or repress (MNT) their transcription.Material and methodsThe cell lines used, URMT and URMax34, derive from MAX-deficient PC12 (rat pheochromocytoma), and carry a pHeBo-MT (empty vector) and a pHeBo-MT-MAX vector (MAX-inducible with Zn+2), respectively. Knockdown of MNT and MLX were achieved with short hairpin RNA constructs (shMNT and shMLX). Proliferation was assessed by cell counting and clonogenic assays; subG0-G1 population was determined by flow cytometry. RNA-seq was performed from two experiments of MNT silencing in URMT and URMax34 cells and confirmed by RT-qPCR. Changes in protein levels were analysed by western blot. Co-immunoprecipitation and proximity ligation assays were used to study protein-protein interactions.Results and discussionsKnocking-down of MNT in UR61 cells resulted in an important decrease in cell proliferation, together with a decrease in both survivin and cyclin A, which are markers of pro-survival and cell proliferation, respectively. DNA content was measured by flow cytometry, revealing an increase in sub-G0 population in shMNT cells. Thus, MNT is required for optimal proliferation of these cells. This is the first evidence of a MAX-independent function of MNT. Then, we extracted RNA from two experiments of MNT silenced and carried out RNA-seq. This resulted in 158 genes whose expression was altered. Cell cycle, DNA replication and DNA repair genes were downregulated upon MNT silencing. However, there were other up-regulated genes like the cell cycle inhibitor CDKN1C (p57). As we confirmed gene regulation by MNT without MAX, we wondered whether it could be working as an heterodimer with MLX or as an homodimer. Co-immunoprecipitation and proximity-ligation assays showed MNT’s ability to form homodimers and heterodimers with MLX. Finally, we carried out MLX knockdown and determined the genes regulated by MNT-MLX or MNT-MNT complexes.ConclusionIn summary, we report novel MAX-independent functions of MNT. In our MAX-deficient model, MNT can be found in homodimers (MNT-MNT) or heterodimers (MNT-MLX) and it supports proliferation and regulates cell cycle and DNA repair genes. This new data about MNT can open new insights into cell biology and tumour development promoted by MYC.

Synovial sarcoma (SS) is driven by a unique chromosomal translocation t(18;X) leading to expression of the SS18-SSXfusion oncoprotein, a transcriptional regulator with both activating and repressing functions. Here we investigated the role of PRAME (Preferentially Expressed Antigen in Melanoma), a protein highly expressed in SS but with a poorly understood function. PRAME is a repressor of retinoic acid (RA) signaling, forming a complex with RA-receptor (RAR) and Enhancer of Zeste Homolog 2 (EZH2). In silico analyses show that expression of PRAME is associated with suppression of RA signaling in SS. The SS18-SSX fusion protein directly targets the PRAME promoter and expression of SS18-SSX and PRAME are positively correlated. As there are no pharmacological inhibitors against PRAME, we used GSK343 for inhibition of EZH2 in combination with all-trans retinoic acid (ATRA) to reconstitute RA signaling. PRAME formed complexes with EZH2 and RAR, while exposure to GSK343 disrupted the PRAME-EZH2 interaction. Combination treatment with GSK343 and ATRA decreased cell proliferation and resulted in cellular senescence. Knockdown of PRAME suppressed the response to ATRA treatment in SS. Our data connect SS18-SSX with RA signaling and the EZH2 complex, providing insights into how this fusion oncoprotein disrupts normal cellular homeostasis.