Role Of MNT Research Articles

A novel role of MNT as a negative regulator of REL and the NF-\u03baB pathway

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.

Abstract A07: MNT-dependent suppression of branched chain amino acid catabolism: A window into the role of MNT:MAX and MNT:MLX complexes in controlling MYC activity

Abstract The MYC family of transcription factors participate in a variety of physiological process and can be oncogenic when dysregulated. A key function of MYC is to reprogram metabolic pathways to meet the biomass needs for both normal the cancer cell proliferation. We previously showed that either too much or too little of the MAX-interacting bHLHZip transcriptional repressor MNT can suppress MYC induced oncogenesis. To begin to investigate the complicated relationship between MYC and MNT, we employed microarray and RNAseq analyses to investigate the gene expression profiles between WT and MNT deficient cells during log phase cell proliferation and during the process of serum stimulated cell cycle entry. Key genes found to be significantly upregulated in MNT deficient cells included ones that promote glycolysis, glutaminolysis, polyamine synthesis, and branched chain amino acid (BCAA) catabolism. Data from metabolomic analysis of MNT-deficient cells was also supportive of elevated glycolysis, glutaminolysis and BCAA catabolism. The high glycolysis and glutaminolysis profiles were similar to MYC-induced metabolic reprograming in tumor cells and T cell activation, but BCAA catabolic activity appeared to be unique to MNT deficient cells. Branched chain α-keto acid dehydrogenase E1α (BCKDH E1α), a subunit of the rate-limit enzyme complex in BCAA catabolism, was found to be one of the most upregulated genes in MNT deficient cells. BCKDH E1α protein was also strongly upregulated in MNT deficient cells, and MNT was found to bind a highly conserved Ebox element in the BCKDH E1αgene and repress its transcription. In contrast to MNT, MYC was found to not regulate BCKDH E1α. Replacing the MNT bHLHZip with the MYC bHLHZip region negated repression and replacing the MYC bHLHZip region with the MNT bHLHZip allowed MYC to activate BCKDH E1α transcription. In contrast to BCKDH E1α, either an engineered Ebox reporter or proximal promoters for specific E-box-containing MYC target genes involved in glycolysis and glutaminolysis that were activated by MYC were repressed by MNT. Protein interaction studies suggest that the differential target gene specificity of MNT compared to MYC is related to the ability of MNT to interact with both MAX and with MLX. Finally, since BCKDH E1α complex was identified as one of the major sources of Reactive Oxygen Species (ROS) in cells and MNT deficient cells have high levels of ROS that sensitizes them to apoptosis (Link et al. PNAS, 2012), we investigated the role of BCKDH E1α in the high ROS in MNT deficient cells and in inhibition of MYC-induced cell transformation. Our findings suggest that elevated BCKDH E1α and BCKDH E1α-induced BCAA catabolism and ROS production significantly contribute to the anti-oncogenic effect of MNT deficiency. Together, our findings suggest that MNT and MYC function as antagonists in some key metabolic pathways, but not in the BCAA catabolism pathway and that the BCAA catabolism pathway may be a potential target for anti-cancer drug development. Citation Format: Guang Yang, Jason M. Link, Peter J. Hurlin, Peter J. Hurlin. MNT-dependent suppression of branched chain amino acid catabolism: A window into the role of MNT:MAX and MNT:MLX complexes in controlling MYC activity. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr A07.

Facile transfer of [2Fe-2S] clusters from the diabetes drug target mitoNEET to an apo-acceptor protein

MitoNEET (mNT) is an outer mitochondrial membrane target of the thiazolidinedione diabetes drugs with a unique fold and a labile [2Fe-2S] cluster. The rare 1-His and 3-Cys coordination of mNT's [2Fe-2S] leads to cluster lability that is strongly dependent on the presence of the single histidine ligand (His87). These properties of mNT are similar to known [2Fe-2S] shuttle proteins. Here we investigated whether mNT is capable of cluster transfer to acceptor protein(s). Facile [2Fe-2S] cluster transfer is observed between oxidized mNT and apo-ferredoxin (a-Fd) using UV-VIS spectroscopy and native-PAGE, as well as with a mitochondrial iron detection assay in cells. The transfer is unidirectional, proceeds to completion, and occurs with a second-order-reaction rate that is comparable to known iron-sulfur transfer proteins. Mutagenesis of His87 with Cys (H87C) inhibits transfer of the [2Fe-2S] clusters to a-Fd. This inhibition is beyond that expected from increased cluster kinetic stability, as the equivalently stable Lys55 to Glu (K55E) mutation did not inhibit transfer. The H87C mutant also failed to transfer its iron to mitochondria in HEK293 cells. The diabetes drug pioglitazone inhibits iron transfer from WT mNT to mitochondria, indicating that pioglitazone affects a specific property, [2Fe-2S] cluster transfer, in the cellular environment. This finding is interesting in light of the role of iron overload in diabetes. Our findings suggest a likely role for mNT in [2Fe-2S] and/or iron transfer to acceptor proteins and support the idea that pioglitazone's antidiabetic mode of action may, in part, be to inhibit transfer of mNT's [2Fe-2S] cluster.

Abstract A65: MicroRNA miR-210 modulates cellular response to hypoxia through the MYC antagonist MNT

Abstract Intratumoral hypoxia is well known as a hallmark of most solid tumors. Here, we studied expression level of about 200 microRNAs in a panel of cancer cell lines from different tissues, and identified a microRNA miR210, its expression was prominently up-regulated by hypoxia. Over expression of miR210 in cancer cell lines partially reversed the hypoxic gene expression signature and allowed cells to escape from hypoxia-induced cell-cycle arrest. We then identified MNT, a known MYC antagonist, as a key miR-210 target. miR-210 directly down-regulated MNT transcript and protein via recognition sites in the transcript 3′ UTR. Consistent with a role for MNT as a miR-210 target, RNA interference-mediated knockdown of MNT phenocopied miR-210 over expression cell cycle effect. Furthermore, loss of MYC abolished the ability of miR-210 to override hypoxia-induced cell-cycle arrest. Thus, miR-210 could function as a modulator in cancer cells in response to hypoxic conditions through Myc network. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A65.