Point mutations in c-Myc uncouple neoplastic transformation from multiple other phenotypes in rat fibroblasts

Deregulation of c-Myc (Myc) occurs in many cancers. In addition to transforming various cell types, Myc also influences additional transformation-associated cellular phenotypes including proliferation, survival, genomic instability, reactive oxygen species production, and metabolism. Although Myc is wild type in most cancers (wtMyc), it occasionally acquires point mutations in certain lymphomas. Some of these mutations confer a survival advantage despite partially attenuating proliferation and transformation. Here, we have evaluated four naturally-occurring or synthetic point mutations of Myc for their ability to affect these phenotypes, as well as to promote genomic instability, to generate reactive oxygen species and to up-regulate aerobic glycolysis and oxidative phosphorylation. Our findings indicate that many of these phenotypes are genetically and functionally independent of one another and are not necessary for transformation. Specifically, the higher rate of glucose metabolism known to be associated with wtMyc deregulation was found to be independent of transformation. One mutation (Q131R) was greatly impaired for nearly all of the studied Myc phenotypes, yet was able to retain some ability to transform. These findings indicate that, while the Myc phenotypes examined here make additive contributions to transformation, none, with the possible exception of increased reliance on extracellular glutamine for survival, are necessary for achieving this state.     

c-Myc is a novel target of cell cycle arrest by honokiol in prostate cancer cells

Honokiol (HNK), a highly promising phytochemical derived from Magnolia officinalis plant, exhibits in vitro and in vivo anticancer activity against prostate cancer but the underlying mechanism is not fully clear. This study was undertaken to delineate the role of c-Myc in anticancer effects of HNK. Exposure of prostate cancer cells to plasma achievable doses of HNK resulted in a marked decrease in levels of total and/or phosphorylated c-Myc protein as well as its mRNA expression. We also observed suppression of c-Myc protein in PC-3 xenografts upon oral HNK administration. Stable overexpression of c-Myc in PC-3 and 22Rv1 cells conferred significant protection against HNK-mediated growth inhibition and G₀-G₁ phase cell cycle arrest. HNK treatment decreased expression of c-Myc downstream targets including Cyclin D1 and Enhancer of Zeste Homolog 2 (EZH2), and these effects were partially restored upon c-Myc overexpression. In addition, PC-3 and DU145 cells with stable knockdown of EZH2 were relatively more sensitive to growth inhibition by HNK compared with control cells. Finally, androgen receptor overexpression abrogated HNK-mediated downregulation of c-Myc and its targets particularly EZH2. The present study indicates that c-Myc, which is often overexpressed in early and late stages of human prostate cancer, is a novel target of prostate cancer growth inhibition by HNK.     

Macrophages expressing triggering receptor expressed on myeloid cells-1 are underrepresented in the human intestine

Triggering receptor expressed on myeloid cells (TREM)-1 is a cell surface molecule on neutrophils and monocytes/macrophages implicated in the amplification of inflammatory responses by enhancing degranulation and secretion of proinflammatory mediators. Macrophages play an important role in the intestinal mucosal immune system, because they are preferentially localized in the subepithelial region. Despite the presence of enormous numbers of bacteria in the colonic mucosa and the close proximity between mucosal macrophages and luminal bacteria, the intestinal mucosa normally displays minimal signs of inflammation. In this study, we show that the resident macrophage population in normal human small and large intestine contains only few TREM-1-expressing macrophages (<10%), whereas the overwhelming majority of monocytes (>90%) and macrophages from lymph nodes or tonsils (>80%) express TREM-1 on the cell surface. These findings were confirmed by FACS analysis and immunostainings of frozen tissue sections. The differential expression of TREM-1 greatly affects the functional capacities of monocytes and tissue macrophages. Although monocytes and macrophages from spleen, lymph nodes, or tonsils show a substantial increase in oxidative burst after TREM-1 cross-linking, no effect is seen in intestinal macrophages. Intriguingly, in contrast to monocytes, intestinal macrophages fail to up-regulate TREM-1 in response to TNF. This refractory state may be induced in intestinal macrophages by the local presence of IL-10 and TGF-beta, because these two immunoregulatory cytokines synergistically down-regulate TREM-1 expression on monocytes in vitro. The absence of TREM-1 expression on lamina propria macrophages is likely to prevent excessive inflammatory reactions, and thus, excessive tissue damage in the intestine.     

Alterations in c-Myc phenotypes resulting from dynamin-related protein 1 (Drp1)-mediated mitochondrial fission

The c-Myc (Myc) oncoprotein regulates numerous phenotypes pertaining to cell mass, survival and metabolism. Glycolysis, oxidative phosphorylation (OXPHOS) and mitochondrial biogenesis are positively controlled by Myc, with myc−/− rat fibroblasts displaying atrophic mitochondria, structural and functional defects in electron transport chain (ETC) components, compromised OXPHOS and ATP depletion. However, while Myc influences mitochondrial structure and function, it is not clear to what extent the reverse is true. To test this, we induced a state of mitochondrial hyper-fission in rat fibroblasts by de-regulating Drp1, a dynamin-like GTPase that participates in the terminal fission process. The mitochondria from these cells showed reduced mass and interconnectivity, a paucity of cristae, a marked reduction in OXPHOS and structural and functional defects in ETC Complexes I and V. High rates of abortive mitochondrial fusion were observed, likely reflecting ongoing, but ultimately futile, attempts to normalize mitochondrial mass. Cellular consequences included reduction of cell volume, ATP depletion and activation of AMP-dependent protein kinase. In response to Myc deregulation, apoptosis was significantly impaired both in the absence and presence of serum, although this could be reversed by increasing ATP levels by pharmacologic means. The current work demonstrates that enforced mitochondrial fission closely recapitulates a state of Myc deficiency and that mitochondrial integrity and function can affect Myc-regulated cellular behaviors. The low intracellular ATP levels that are frequently seen in some tumors as a result of inadequate vascular perfusion could favor tumor survival by countering the pro-apoptotic tendencies of Myc overexpression.     

The c-Myc target gene Rcl (C6orf108) encodes a novel enzyme, deoxynucleoside 5'-monophosphate N-glycosidase

RCL is a c-Myc target with tumorigenic potential. Genome annotation predicted that RCL belonged to the N-deoxyribosyltransferase family. However, its putative relationship to this class of enzymes did not lead to its precise biochemical function. The purified native or N-terminal His-tagged recombinant rat RCL protein expressed in Escherichia coli exhibits the same enzyme activity, deoxynucleoside 5'-monophosphate N-glycosidase, never before described. dGMP appears to be the best substrate. RCL opens a new route in the nucleotide catabolic pathways by cleaving the N-glycosidic bond of deoxynucleoside 5'-monophosphates to yield two reaction products, deoxyribose 5-phosphate and purine or pyrimidine base. Biochemical studies show marked differences in the terms of the structure and catalytic mechanism between RCL and of its closest enzyme family neighbor, N-deoxyribosyltransferase. The reaction products of this novel enzyme activity have been implicated in purine or pyrimidine salvage, glycolysis, and angiogenesis, and hence are all highly relevant for tumorigenesis.     

c-Myc is a critical target for c/EBPalpha in granulopoiesis

CCAAT/enhancer binding protein alpha (C/EBPalpha) is an integral factor in the granulocytic developmental pathway, as myeloblasts from C/EBPalpha-null mice exhibit an early block in differentiation. Since mice deficient for known C/EBPalpha target genes do not exhibit the same block in granulocyte maturation, we sought to identify additional C/EBPalpha target genes essential for myeloid cell development. To identify such genes, we used both representational difference analysis and oligonucleotide array analysis with RNA derived from a C/EBPalpha-inducible myeloid cell line. From each of these independent screens, we identified c-Myc as a C/EBPalpha negatively regulated gene. We mapped an E2F binding site in the c-Myc promoter as the cis-acting element critical for C/EBPalpha negative regulation. The identification of c-Myc as a C/EBPalpha target gene is intriguing, as it has been previously shown that down-regulation of c-Myc can induce myeloid differentiation. Here we show that stable expression of c-Myc from an exogenous promoter not responsive to C/EBPalpha-mediated down-regulation forces myeloblasts to remain in an undifferentiated state. Therefore, C/EBPalpha negative regulation of c-Myc is critical for allowing early myeloid precursors to enter a differentiation pathway. This is the first report to demonstrate that C/EBPalpha directly affects the level of c-Myc expression and, thus, the decision of myeloid blasts to enter into the granulocytic differentiation pathway.     

Oxidation resistance 1 is a novel senolytic target

The selective depletion of senescent cells (SCs) by small molecules, termed senolytic agents, is a promising therapeutic approach for treating age‐related diseases and chemotherapy‐ and radiotherapy‐induced side effects. Piperlongumine (PL) was recently identified as a novel senolytic agent. However, its mechanism of action and molecular targets in SCs was unknown and thus was investigated. Specifically, we used a PL‐based chemical probe to pull‐down PL‐binding proteins from live cells and then mass spectrometry‐based proteomic analysis to identify potential molecular targets of PL in SCs. One prominent target was oxidation resistance 1 (OXR1), an important antioxidant protein that regulates the expression of a variety of antioxidant enzymes. We found that OXR1 was upregulated in senescent human WI38 fibroblasts. PL bound to OXR1 directly and induced its degradation through the ubiquitin‐proteasome system in an SC‐specific manner. The knockdown of OXR1 expression by RNA interference significantly increased the production of reactive oxygen species in SCs in conjunction with the downregulation of antioxidant enzymes such as heme oxygenase 1, glutathione peroxidase 2, and catalase, but these effects were much less significant when OXR1 was knocked down in non‐SCs. More importantly, knocking down OXR1 selectively induced apoptosis in SCs and sensitized the cells to oxidative stress caused by hydrogen peroxide. These findings provide new insights into the mechanism by which SCs are highly resistant to oxidative stress and suggest that OXR1 is a novel senolytic target that can be further exploited for the development of new senolytic agents.     

PAP-1, a novel target protein of phosphorylation by pim-1 kinase.

Protooncogene, pim-1, has been reported to be a predisposition for lymphomagenesis along with myc, and its protein product, Pim-1, has been shown to be a serine/threonine protein kinase, whose activity is involved in proliferation and differentiation of blood cells. The signal transduction pathways neither to nor from Pim-1, however, have been clarified. We have cloned a cDNA encoding a novel Pim-1 binding protein, PAP-1, comprising 213 amino acids with a basic amino-acid cluster near the C-terminus. PAP-1 was colocalized with Pim-1 in human HeLa cell nuclei. The in vitro binding assays using GST fusion proteins of the wild-type and various deletion mutants revealed that the whole molecule of Pim-1 is required for the binding activity to PAP-1 and that Pim-1 binds to the region from amino-acid numbers 1-147 of PAP-1, or to two segments in the region. The association of PAP-1 with Pim-1 was also shown in vivo in transfected cells. Furthermore, PAP-1 was phosphorylated in vitro by Pim-1, but not a kinase-negative Pim-1 mutant. The two serine residues of PAP-1 at amino acids 204 and 206 near the C-terminus were phosphorylated by Pim-1. PAP-1 is thus thought to be a target protein for Pim-1 kinase.     

mRTVP-1, a novel p53 target gene with proapoptotic activities

We identified a novel mouse gene, mRTVP-1, as a p53 target gene using differential display PCR and extensive promoter analysis. The mRTVP-1 protein has 255 amino acids and differs from the human RTVP-1 (hRTVP-1) protein by two short in-frame deletions of two and nine amino acids. RTVP-1 mRNA was induced in multiple cancer cell lines by adenovirus-mediated delivery of p53 and by gamma irradiation or doxorubicin both in the presence and in the absence of endogenous p53. Analysis of RTVP-1 expression in nontransformed and transformed cells further supported p53-independent gene regulation. Using luciferase reporter and electrophoretic mobility shift assays we identified a p53 binding site within intron 1 of the mRTVP-1 gene. Overexpression of mRTVP-1 or hRTVP-1 induced apoptosis in multiple cancer cell lines including prostate cancer cell lines 148-1PA, 178-2BMA, PC-3, TSU-Pr1, and LNCaP, a human lung cancer cell line, H1299, and two isogenic human colon cancer cell lines, HCT116 p53(+/+) and HCT116 p53(-/-), as demonstrated by annexin V positivity, phase-contrast microscopy, and in selected cases 4',6'-diamidino-2-phenylindole staining and DNA fragmentation. Deletion of the signal peptide from the N terminus of RTVP-1 reduced its apoptotic activities, suggesting that a secreted and soluble form of RTVP-1 may mediate, in part, its proapoptotic activities.