Mxi1 Expression Research Articles

Exploring the relationship between red blood cell levels and emotional regulation through the miR191-Riok3-Mxi1 pathway

ObjectiveTo assess emotional fluctuations, physical and mental health status, and indicators closely related to red blood cells, such as RIO kinase 3 (Riok3), MAX interactor 1 (Mxi1), and microRNA 191 (miR191), in participants with different levels of red blood cells. MethodsParticipants who underwent physical examinations at Dongfang Hospital between April and October 2019 were divided into healthy, blood deficiency, and anemia groups (30 individuals in the healthy and blood deficiency group respectively, and 13 in the anemia group). The physical and mental conditions of the participants were evaluated through questionnaires, and emotional fluctuations were assessed through an emotion-inducing experiment, in which participants watched video segments designed to induce specific emotions. Relative expression levels of miR191, Riok3, and Mxi1 from venous blood samples were also determined. ResultsThe main psychological factors identified in the anemia and blood deficiency groups were obsessive-compulsive symptoms, depression, anxiety, and other negative emotions. Relative gene expression levels indicated that miR191 was upregulated and Riok3 and Mxi1 were downregulated in both the blood deficiency and anemia groups. Regarding the emotional score of disgust on video stage, the main effect was significant (F = 335.58, P < .001), which showed that watching the three videos caused participants to have a dominant emotion, and there is a difference on group (F = 5.35, P = .01), with higher disgust scores in the anemia and blood deficiency groups. The symptoms of blood deficiency and anemia, such as weakness in limbs were significantly negatively correlated with Riok3 and Mxi1 expression (r = −0.38 and −0.31 respectively), but was significantly positively correlated with miR191 expression (r = 0.29). ConclusionWe determined that a close relationship exists between red blood cell levels and emotional status. Our findings suggest that individuals with anemia and blood deficiency are more likely to experience psychological problems and negative emotions, particularly disgust. We also demonstrate that emotional regulation is related to mir191-Riok3-Mxi1 pathway activity, identifying these pathway components are potential targets for genetic therapies in combination with psychological therapy.

FTO Inhibition Enhances the Antitumor Effect of Temozolomide by Targeting MYC-miR-155/23a Cluster-MXI1 Feedback Circuit in Glioma

Malignant glioma constitutes one of the fatal primary brain tumors in adults. Such poor prognosis calls for a better understanding of cancer-related signaling pathways of this disease. Here we elucidate a MYC-miRNA-MXI1 feedback loop that regulates proliferation and tumorigenesis in glioma. MYC suppressed MXI1 expression via microRNA-155 (miR-155) and the microRNA-23a∼27a∼24-2 cluster (miR-23a cluster), whereas MXI1, in turn, inhibited MYC expression by binding to its promoter. Overexpression of miR-155 and the miR-23a cluster promoted tumorigenesis in U87 glioma cells. Furthermore, fat mass and obesity-associated protein (FTO), an N6-methyladenosine (m6A) RNA demethylase, regulated the loop by targeting MYC. The ethyl ester form of meclofenamic acid (MA2) inhibited FTO and enhanced the effect of the chemotherapy drug temozolomide on suppressing proliferation of glioma cells and negatively regulated the loop. These data collectively highlight a key regulatory circuit in glioma and provide potential targets for clinical treatment. SIGNIFICANCE: These findings elucidate a novel feedback loop that regulates proliferation in glioma and can be targeted via inhibition of FTO to enhance the efficacy of temozolomide.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

S6K1 phosphorylation-dependent degradation of Mxi1 by β-Trcp ubiquitin ligase promotes Myc activation and radioresistance in lung cancer.

Rationale: Mxi1 is regarded as a potential tumor suppressor protein that antagonizes the transcriptional activity of proto-oncogene Myc. However, the clinical significances and underlying mechanisms by which Mxi1 is regulated in lung cancer remain poorly understood.Methods: Mass spectrometry analysis and immunoprecipitation assay were utilized to detect the protein-protein interaction. The phosphorylation of Mxi1 was evaluated by in vitro kinase assays. Poly-ubiquitination of Mxi1 was examined by in vivo ubiquitination assay. Lung cancer cells stably expressing wild-type Mxi1 or Mxi1-S160A were used for functional analyses. The expression levels of Mxi1 and S6K1 were determined by immunohistochemistry in lung cancer tissues and adjacent normal lung tissues.Results: We found that Mxi1 is downregulated and correlated with poor prognosis in lung cancer. Using tandem affinity purification technology, we provided evidence that β-Trcp E3 ubiquitin ligase interacts with and promotes the ubiquitination and degradation of Mxi1. Furthermore, we demonstrated that Mxi1 is phosphorylated at S160 site by the protein kinase S6K1 and subsequently degraded via the ubiquitin ligase β-Trcp. Moreover, a phosphorylation mutant form of Mxi1 (Mxi1-S160A), which cannot be degraded by S6K1 and β-Trcp, is much more stable and efficient in suppressing the transcriptional activity of Myc and radioresistance in lung cancer cells. More importantly, a strong inverse correlation between S6K1 and Mxi1 expression was observed in human lung cancer tissues.Conclusion: Our findings not only establish a crosstalk between the mTOR/S6K1 signaling pathway and Myc activation, but also suggest that targeting S6K1/Mxi1 pathway is a promising therapeutic strategy for the treatment of lung cancer.

Interaction of RNA-binding protein HuR and miR-466i regulates GM-CSF expression

Granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by T helper 17 (Th17) cells plays an essential role in autoimmune diseases. Transcriptional regulation of Th17 cell differentiation has been extensively studied, but post-transcriptional regulation of Th17 cell differentiation has remained less well characterized. The RNA-binding protein HuR functions to promote the stability of target mRNAs via binding the AU-rich elements of the 3′ untranslated region (3′UTR) of numerous pro-inflammatory cytokines including IL-4, IL-13, IL-17 and TNF-α. However, whether HuR regulates GM-CSF expression in Th17 cells has not been fully investigated. Here we showed that HuR conditional knockout (KO) Th17 cells have decreased GM-CSF mRNA in comparison with wild-type (WT) Th17 cells, and that HuR binds directly to GM-CSF mRNA 3′UTR. Interestingly, HuR deficiency increased the levels of certain microRNA expression in Th17 cells; for example, miR-466i functioned to mediate GM-CSF and IL-17 mRNA decay, which was confirmed by in vitro luciferase assay. Furthermore, we found that HuR promoted Mxi1 expression to inhibit certain miRNA expression. Taken together, these findings indicate that interaction of HuR and miR-466i orchestrates GM-CSF expression in Th17 cells.

Abstract B32: The role of Exon 0 in mediating Mxi0 activity in neuroblastoma

Abstract Background: Neuroblastoma is the most common extracranial malignancy of childhood. The Myc family of proteins regulates cell growth and proliferation and has been implicated in the etiology of many cancers. MYCN amplified neuroblastoma is associated with a poor prognosis. Investigating specific tumor pathways will further our understanding of neuroblastoma pathogenesis and lead to future therapeutic options. Mxi1 is a member of the MAD family that inhibits N-Myc activity. Mxi0 is an alternatively spliced variant of Mxi1 with a different first exon (Exon 0) whose function has not been determined. These proteins appear to have differential functions in neuroblastoma pathogenesis. Objective: Elucidate the impact of Mxi1 and Mxi0 expression on neuroblastoma physiology and determine the role of Exon 0 in the function of Mxi0. Design: We created neuroblastoma cell lines with inducible expression of Mxi1 and Mxi0 to tightly control expression. Cell proliferation and survival were quantified using BrdU and MTT assays. Chemosensitivity was assessed by treating cells with doxorubicin or etoposide after induction of Mxi1 or Mxi0 expression. Cell viability was then measured by by MTT assay. To help examine the role of Exon 0, we utilized GFP tagged constructs of Mxi1, Mxi0, and Exon 0. These proteins were expressed in 293T cells and subcellular localization of Mxi1, Mxi0, and Exon 0 proteins was then detected by immunofluorescence. To assess the role of nuclear export in cell localization, cells were treated with leptomycin B. Results: Overexpression of Mxi1 inhibits N-Myc mediated cell proliferation. Conversely, overexpression of Mxi0 in neuroblastoma cell lines leads to enhanced proliferation, suggesting that Mxi0 has a counter-regulatory role to that of Mxi1. Compared with Mxi1, expression of Mxi0 results in cells becoming more chemoresistant. Examination of Mxi1 and Mxi0 subcellular location reveals that Mxi1 resides in the nucleus while Mxi0 is found primarily in the cytoplasm. Exon 0 alone also is found in the cytoplasm, indicating that this differential localization is determined by the presence of Exon 0. Finally, treatment with leptomycin B resulted in accumulation of Mxi0 in the nucleus, suggesting that it may cycle in and out of the nucleus in response to appropriate signals. Conclusions: Overexpression of Mxi1 in neuroblastoma cell lines leads to inhibition of N-Myc-mediated cell proliferation while Mxi0 appears to promote cell growth. Mxi1 expression enhances chemosensitivity of neuroblastoma cells, while Mxi0 has the opposite effect. Exon 0 directs the cytoplasmic localization of Mxi0 and may play an important role in its differential function. A better understanding of how the interaction between Mxi1 and Mxi0 affects neuroblastoma physiology and how Exon 0 imparts the differential function of Mxi0 may aid in developing more effective targeted therapies to improve outcomes in children with neuroblastoma. Citation Format: Stephen J. Kirchner, James T. Bartram, D. Christian Ellis, Daniel S. Wechsler, Michael B. Armstrong. The role of Exon 0 in mediating Mxi0 activity in neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B32.

CXCL10 expression induced by Mxi1 inactivation induces mesangial cell apoptosis in mouse Habu nephritis

MAX interactor 1 (Mxi1) proteins are c-myc antagonists that primarily exert their biological functions by inhibiting Myc-dependent gene transcription. In this study, Mxi1−/− mice were used to generate a model of mesangial proliferative glomerulonephritis for the first time. In the present study, we demonstrated that Mxi1−/− mice exhibited a more typical and severe pathological phenotype, which was displayed primarily as a noticeable dissolution phenotype with a higher proportion of apoptotic cells and higher chemokine CXCL10 expression during the early days of modeling, compared with wild-type mice. Additionally, we determined that IRF3-mediated TLR4 signaling was likely involved in regulating CXCL10 expression, which might participate in the mesangial dissolution process. We also found increases in CXCL10 expression, caspase 3 activation, and the proportion of apoptotic cells when Mxi1 expression was inhibited in mouse mesangial cells. Furthermore, the proportion of apoptotic cells decreased after inhibiting CXCL10 expression. Therefore, we concluded that the mesangial cell apoptosis observed in this mesangial proliferative glomerulonephritis model was related to CXCL10 expression induced by Mxi1 inactivation. This finding provides a new theoretical basis for the mechanism of mesangial proliferative glomerulonephritis progression and reveals potential intervention targets for the early treatment of this disease.

A Systems Approach Identifies Essential FOXO3 Functions in Erythroblast Enucleation Process

Elucidating mechanisms that regulate terminal erythroblast maturation may serve to improve the treatment of anemias as well as the generation of red blood cells in vitro. Key steps in red blood cell maturation are chromatin condensation and expulsion of the nucleus (or enucleation) from late-stage erythroblasts. The transcriptional program that coordinates these steps is poorly defined. The expression and transcriptional activity of FOXO3 increase with terminal erythroblast maturation suggesting a potential function for FOXO3 in this process. Using comparative transcriptomic analysis of primary freshly isolated wild type and Foxo3-/- erythroblasts at distinct stages of maturation, we found that many genes involved in DNA packaging-related pathways were highly downregulated in Foxo3-/- erythroblasts, raising the possibility that FOXO3 might be involved in regulating chromatin condensation and/or the enucleation process. Foxo3-/- enucleating cells were also significantly fewer than wild type bone marrow erythroblasts according to in vivo DNA staining by DRAQ5. qRT-PCR analysis confirmed the reduced expression of genes implicated in the control of chromatin condensation and/or enucleation including Mxi1, Riok3, Trim58, Rac GTPase I and II at most if not all stages of maturation in Foxo3-/- erythroblasts. Immunoprecipitation of endogenous FOXO3 in wild type but not in Foxo3-/- bone marrow erythroblasts recovered the regulatory regions of both Mxi1 and Riok3 in vivo suggesting that FOXO3 controls directly the expression of these genes in erythroblasts. To investigate more directly the potential impact of loss of FOXO3 on the enucleation process, we examined bone marrow erythroblasts at high magnification by confocal fluorescence microscopy. Wild-type enucleating erythroblasts were identified by a gap in the bright TER119 membrane staining and displayed a dumbbell-shaped nucleus, with a neck located at the TER119 sorting boundary of the nascent reticulocyte, so that the cells used a single direction for nuclear extrusion. However, Foxo3 mutant erythroblasts exhibited multiple nuclear necks accompanied by multiple sorting boundaries with each lobe extruding in a different direction away from the nascent reticulocyte, suggesting defective polarization of Foxo3 mutant erythroblasts during enucleation. As a result 48% of the Foxo3-/-enucleating erythroblasts displayed abnormal enucleation morphologies. Next using imaging flow cytometry we further characterized the potential enucleation defect in Foxo3-/- erythroblasts. Segregation of TER119+ erythroblasts according to their cell body and nuclear size into distinct stages of maturation (pro-, basophilic, polychromatic and orthochromatic) showed a relative increase in late erythroid precursors in Foxo3-/- bone marrow. It also revealed that the Foxo3 mutant erythroblasts were specifically accumulated at the orthochromatic stage of maturation. The percentage of orthochromatic erythroblasts containing an asymmetric nucleus was quantified using the delta centroid, a measure of the distance between the center of the cell body and center of the DRAQ5-stained nucleus. This analysis revealed a lower percentage of asymmetric enucleating erythroblasts in the Foxo3-/- as compared to wild-type bone marrow. Using this approach lobular nuclei were distinguished from circular nuclei and demonstrated that odd nuclei were present with remarkably greater frequency in Foxo3-/- than in wild type orthochromatic erythroblasts. These combined analyses support the notion that FOXO3 is critical for the erythroblast enucleation process and may be implicated in polarization of erythroblasts leading to enucleation. The direct ability of FOXO3 to rescue the defective Foxo3 mutant erythroblast enucleation was shown by ectopic expression MSCV-IRES-GFP (MIG)-FOXO3 as compared to empty control vector in wild type and Foxo3-/- bone marrow-derived erythroid progenitors. Restoration of FOXO3 in GFP-positive Foxo3-/-erythroblasts rescued the enucleation rate to similar levels as controls after 72 hours of retroviral transduction and significantly enhanced the levels of expression of Mxi1, Riok3 and Trim58 in Foxo3-/-erythroblasts. Collectively, these findings show that FOXO3 is an essential component of the transcriptional program that regulates terminal erythroblast maturation and is implicated in the enucleation process. DisclosuresNo relevant conflicts of interest to declare.

Abstract B37: The impact of modulating Mxi1 and Mxi0 expression on N-Myc-mediated neuroblastoma tumor pathogenesis and chemosensitivity

Abstract Background: Neuroblastoma is the most common extracranial malignancy of childhood. Myc family proteins regulate cell growth and proliferation in response to mitogenic stimulation, and Myc proteins are implicated in the etiology of many cancers. MYCN amplified neuroblastoma carries a poor overall survival with limited therapeutic options. Investigating specific tumor pathways will further our understanding of neuroblastoma pathogenesis and lead to future therapeutic options for children with this disease. Mxi1 is a member of the MAD family of proteins that inhibit N-Myc function. Mxi0 is an alternatively-spliced variant of Mxi1 whose function has not been determined. We hypothesize that Mxi1 and Mxi0 expression levels affect N-Myc-dependent neuroblastoma cell growth. Objective: To determine the impact of modulating Mxi1 and Mxi0 expression on N-Myc-dependent neuroblastoma cell proliferation and survival. Design: We expressed Mxi1 and Mxi0 in SHEP neuroblastoma cells and SHEP cells stably transfected to express high levels of MYCN (SHEP/MYCN). We also utilized native neuroblastoma cell lines with inducible expression of Mxi1 and Mxi0. Cell proliferation and survival were quantified using BrdU and MTT assays, respectively. Apoptosis was measured by propidium iodide staining and caspase-3 immunohistochemistry. Cellular localization of Mxi1 and Mxi0 proteins was detected by immunofluorescence. Results: Overexpression of Mxi1 inhibits N-Myc mediated cell proliferation. Additionally, in the absence of N-Myc, Mxi1 overexpression independently inhibits cell proliferation and induces cell apoptosis. Conversely, overexpression of Mxi0 in neuroblastoma cell lines leads to enhanced proliferation, suggesting that Mxi0 has a counter-regulatory role to that of Mxi1. Expression of Mxi1 increased sensitivity of the neuroblastoma cells to doxorubicin, while higher levels of Mxi0 made the cells more chemoresistant. Finally, examination of Mxi1 and Mxi0 cellular location reveals that Mxi1 resides in the nucleus while Mxi0 is found primarily in the cytoplasm. Conclusions: Overexpression of Mxi1 in neuroblastoma cell lines leads to inhibition of N-Myc-mediated cell proliferation while Mxi0 appears to promote cell growth. Mxi1 expression enhanced chemosensitivity of neuroblastoma cells, while Mxi0 had the converse effect. A better understanding of the interaction between Mxi1 and Mxi0 and how the balance of these proteins affect neuroblastoma physiology may aid in developing more effective targeted therapies to improve outcomes in children with neuroblastoma. Citation Format: D. Christian Ellis, R. Scott Widemon, Daniel S. Wechsler, Michael B. Armstrong. The impact of modulating Mxi1 and Mxi0 expression on N-Myc-mediated neuroblastoma tumor pathogenesis and chemosensitivity. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B37.

N-Myc Differentially Regulates Expression of MXI1 Isoforms in Neuroblastoma

Amplification of the MYCN proto-oncogene is associated with a poor prognosis in patients with metastatic neuroblastoma (NB). MYCN encodes the N-Myc protein, a transcriptional regulator that dimerizes with the Max transcription factor, binds to E-box DNA sequences, and regulates genes involved in cell growth and apoptosis. Overexpression of N-Myc leads to transcriptional activation and an increase in NB cell proliferation. Mxi1, a member of the Myc family of transcriptional regulators, also binds to Max. However, Mxi1 is a transcriptional repressor and inhibits proliferation of NB cells, suggesting that Mxi1 functions as an N-Myc antagonist. Our laboratory previously identified Mxi1-0, an alternatively transcribed Mxi1 isoform. Mxi1-0 has properties distinct from those of Mxi1; in contrast to Mxi1, Mxi1-0 is unable to suppress c-Myc-dependent transcription. We now show that Mxi1-0 expression increases in response to MYCN overexpression in NB cells, with a positive correlation between MYCN and MXI1-0 RNA levels. We also show that N-Myc expression differentially regulates the MXI1 and MXI1-0 promoters: Increased MYCN expression suppresses MXI1 promoter activity while enhancing transcription through the MXI1-0 promoter. Finally, induction of Mxi1-0 leads to increased proliferation, whereas expression of Mxi1 inhibits cell growth, indicating differential roles for these two proteins. These data suggest that N-Myc differentially regulates the expression of MXI1 and MXI1-0 and can alter the balance between the two transcription factors. Furthermore, MXI1-0 appears to be a downstream target of MYCN-dependent signaling pathways and may contribute to N-Myc-dependent cell growth and proliferation.

Change of MAX Interactor 1 Expression in an Anti-Thy1 Nephritis Model and its Effect on Mesangial Cell Proliferation

Background/Aims: During the disease process of mesangial proliferative glomerulonephritis, the expression of various factors that influence mesangial proliferation is altered. MAX interactor 1 (Mxi1) antagonizes the transcription factor Myc and is believed to be a tumor suppressor. However, no studies have investigated its effect on mesangial cell proliferation. Methods: To investigate the effect of Mxi1 on renal mesangial cell proliferation, we established a classic rat anti-Thy1 mesangial proliferative glomerulonephritis model. Mesangial proliferation was estimated by immunohistochemical analysis of Ki67. Mxi1 expression at each time point was assessed by real-time RT-PCR and Western blot analyses. Furthermore, we altered the expression level of Mxi1 by a plasmid and siRNA to detect its effect on rat mesangial cell proliferation in vitro. Results: Mxi1 expression decreased significantly during the proliferative period of anti-Thy1 nephritis model and then gradually increased as proliferation declined, indicating that Mxi1 may be linked to mesangial cell proliferation. Upregulation of Mxi1 expression via plasmid transfection in vitro reduced the expression of the positive-acting cell cycle regulatory proteins cyclin B1, cyclin D1, cyclin E, CDC2 and CDK2; significantly reduced mesangial cell proliferation; reduced the percentage of S phase cells; and increased the percentage of G2/M phase cells. Inhibition of Mxi1 expression by siRNA in vitro produced the opposite effects: increased expression of cyclin B1, cyclin D1, cyclin E, CDC2 and CDK2; markedly increased cell proliferation; higher percentage of S phase cells; and dramatically lower percentage of G2/M phase cells. Transcription factor c-myc protein expression showed no obvious difference after Mxi1 plasmid and siRNA transfection. The expressions of cell cycle regulatory proteins mentioned above were negative correlated with Mxi1 expression in anti-Thy1 nephritis model. Conclusion: These results suggest that Mxi1 expression levels were inversely correlated with proliferation in anti-Thy1 nephritis rats and it may influence cell cycle progression and thus the rate of mesangial cell proliferation by regulating the expression of c-myc target cell cycle regulatory proteins.

Mxi1-SRalpha: a novel Mxi1 isoform with enhanced transcriptional repression potential.

Mxi1 belongs to the Myc/Max/Mad network of proteins that have been implicated in the control of multiple aspects of cellular behavior. Previously, we had reported that the mouse mxi1 gene gives rise to two distinct transcript forms that can encode proteins with dramatically different functional abilities. The Mxi1-SR protein (here termed Mxi1-SRbeta) can interact with Sin3/histone deacetylase and function as a potent transcriptional repressor and growth suppressor, while the Mxi1-WR protein lacks these activities. Here, we describe a new mxi1-derived transcript form (termed mxi1-SRalpha) whose expression is governed by its own promoter, resulting in a spatiotemporally distinct expression profile from that of the highly related mxi1-SRbeta form. Moreover, the Mxi1-SRalpha protein product, with its unique Sin3 interacting domain, has a greater affinity than its Mxi1-SRbeta counterpart for the Sin3 adapter proteins as well as an enhanced potential for transcriptional repression in transient reporter assays. Our identification of this novel Mxi1 isoform that results from alternative 5' exon usage adds an additional layer of complexity to the Mad/Mxi1 family. In addition, our findings warrant re-evaluation of mxi1 expression patterns on the cellular level and its status in human cancer samples, with a renewed focus on the distinct isoforms.

Autorepression of c-myc requires both initiator and E2F-binding site elements and cooperation with the p107 gene product.

Myc proteins are transcriptional activators, but also repress transcription through initiator (Inr) elements. Repression requires the conserved Myc Box II, but the cis-acting element(s) required for c-myc autorepression have eluded definition. Since the gene has a candidate Inr at the P2 promoter, we tested whether Myc autorepression operates through the Inr/BoxII mechanism. Overexpression of c-Myc but not a Box II deletion mutation represses both c-myc P2 reporter genes and endogenous c-myc, as does Mxi1 expression. Only 45 nucleotides surrounding the P2 start suffice to mediate autorepression, but Myc and Mxi1 also downregulate P2 Inr mutations, suggesting other core promoter sequence requirements for autorepression. We tested the importance of conserved E2F sites, based on known Myc interaction with the pRb-related p107 and on the transrepressive effects of Rb family proteins. Myc, Mxi1, and p107 repress c-myc somewhat less well in the absence of E2F binding sites, while an E2F+Inr double mutation is not repressed at all by these gene products. Further, Myc repression at the c-myc P2 core promoter is augmented by p107, but not by pRb or p130, nor by p107 lacking the conserved pocket domain. Our data suggest that Myc autorepression requires both the c-myc Inr and E2F sites in cis, as well as p107 in trans. Consistent with this, we found that retrovirally transduced c-Myc cannot downregulate endogenous c-myc in p107-null fibroblasts, and show evidence that both Myc and p107 are present in a complex assembled at the c-myc P2 core promoter.

Mxi1, a Myc antagonist, suppresses proliferation of DU145 human prostate cells

Mxi1, an antagonist of c-Myc, maps to human chromosome 10q24-q25, a region altered in a substantial fraction of prostate tumors. Mice deficient for Mxi1 exhibit significant prostate hyperplasia. We studied the ability of Mxi1 to act as a growth suppressor in prostate tumor cells. We infected DU145 prostate carcinoma cells with an Mxi1-expressing adenovirus (AdMxi1) in vitro, and measured Mxi1 expression, cell proliferation, soft agar colony formation, and cell cycle distribution. To explore mechanisms of Mxi1-induced growth arrest, we performed gene expression analysis. AdMxi1 infection resulted in reduced cell proliferation, reduced soft agar colony formation, and a higher proportion of cells in the G(2)/M phase of the cell cycle. This G(2)/M growth arrest was associated with elevated levels of cyclin B, and reduced levels of c-MYC and MDM2. The ability of AdMxi1 to suppress prostate tumor cell proliferation supports a role for Mxi1 loss in the pathogenesis of a subset of human prostate cancers. Prostate 47:194-204, 2001.

Expression of MXI1, a Myc Antagonist, Is Regulated by Sp1 and AP2

MXI1, a member of the MAD family of Myc antagonists, encodes a transcription factor whose expression must be tightly regulated to maintain normal cell growth and differentiation. To more closely investigate the transcriptional regulation of the human MXI1 gene, we have cloned and characterized the MXI1 promoter. After clarification of the 5'- and 3'-untranslated regions of the cDNA (indicating that the true length of the MXI1 transcript is 2643 base pairs), we identified two transcription initiation sites. We subsequently isolated the MXI1 promoter, which is GC-rich and lacks a TATA box. Although it contains at least six potential initiator sequences, functional studies indicate the proximal two initiator sequences in combination with nearby Sp1 and MED-1 sites together account for virtually all promoter activity. We also demonstrate that MXI1 promoter activity is repressed by high levels of AP2. These studies provide further insight into the complex regulatory mechanisms governing MXI1 gene expression and its role in cellular differentiation and tumor suppression.

Suppression of Myc, but not E1a, transformation activity by Max-associated proteins, Mad and Mxi1.

Mad and Mxi1, two members of the Myc-related basic-region helix-loop-helix/leucine-zipper family of proteins, associate directly with Max to form sequence-specific DNA binding heterodimers that are transactivation-incompetent. Mad-Max complexes have been shown to exert a strong repressive effect on Myc-induced transactivation, perhaps through the competitive occupation of common promoter binding sites also recognized by active Myc-Max heterodimers. To place these recent biochemical observations in a biological context, mad and mxi1 expression vectors were tested for their ability to influence Myc transformation activity in the rat embryo fibroblast cooperation assay. Addition of an equimolar amount of mad or mxi1 expression vector to mouse c-myc/ras cotransfections resulted in a dramatic reduction in both the number of foci generated and the severity of the malignant phenotype. Myc-specific suppression by Mad and Mxi1 was demonstrated by their ability to affect c- and N-myc-, but not ela-, induced transformation. In contrast, mad and mxi1 expression constructs bearing deletions in the basic region exerted only mild repressive effects on Myc transformation activity, suggesting that occupation of common DNA binding sites by transactivation-incompetent Mad-Max or Mxi1-Max complexes appears to play a more dominant role in this suppression than titration of limited intracellular pools of Max away from active Myc-Max complexes. Thus, these biological data support a current model for regulation of Myc function in which relative intracellular levels of Mad and Mxi1 in comparison to those of Myc may determine the degree of activation of Myc-responsive growth pathways.