diminutive
The transactivation of TCF target genes induced by Wnt pathway mutations constitutes the primary transforming event in colorectal cancer (CRC). Disruption of ß-catenin/TCF-4 activity in CRC cells induces a rapid G1 arrest and blocks a genetic program that is physiologically active in the proliferative compartment of colon crypts. Coincidently, an intestinal differentiation program is induced. The TCF-4 target gene c-MYC plays a central role in this switch by direct repression of the p21CIP1/WAF1 promoter. Following disruption of ß-catenin/TCF-4 activity, the decreased expression of c-MYC releases p21CIP1/WAF1 transcription, which in turn mediates G1 arrest and differentiation. Thus, the ß-catenin/TCF-4 complex constitutes the master switch that controls proliferation versus differentiation in healthy and malignant intestinal epithelial cells (van de Wetering, 2002).
c-MYC plays a central role in the proliferative capacity of many cancers, including CRC. tHE data imply that c-MYC blocks the expression of the cell cycle inhibitor p21CIP1/WAF1. The region responsible for p21CIP1/WAF1 regulation has been mapped to a 200 bp fragment of the proximal promoter.
The presence of MIZ-1 and c-MYC on this promoter suggests that c-MYC-mediated repression of p21CIP1/WAF1 occurs by a mechanism resembling c-MYC control of p15INK4b, i.e., through preventing promoter activation by the transcription factor MIZ-1. Decreased expression of c-MYC would allow MIZ-1 to activate p21CIP1/WAF1 transcription. The complementarity in the expression of c-MYC and p21CIP1/WAF1 in the intestine supports this mechanism (van de Wetering, 2002).
The carboxyl terminus of c-Myc, containing the basic region (B) and helix-loop-helix/leucine zipper
(HLH/LZ) domain, is necessary and sufficient for sequence-specific DNA binding and
heterodimerization with Max. The amino terminus, containing two highly conserved regions
termed Myc box (Mb) I and II, is necessary for transcriptional activation and repression. Both
the transactivation domain (TAD) and the BHLH/LZ domain are necessary for
biological activity. The c-MycS proteins arise from a
leaking scanning mechanism and initiate at two closely spaced downstream AUG codons, yielding
c-Myc proteins lacking ~100 amino-terminal amino acids, including the highly conserved MbI region.
Synthesis of c-MycS increases to levels comparable to c-Myc2 during rapid cell growth, and
constitutively high levels of c-MycS synthesis are found in some tumor cell lines. Transcriptional activation by c-Myc through specific E box elements is thought to be essential for its biological role.
However, c-MycS is unable to activate transcription through these elements and yet retains the ability to stimulate
proliferation, induce anchorage-independent growth, and induce apoptosis. In addition, c-MycS retains the ability to
repress transcription of several specific promoters. Furthermore, c-MycS can rescue the c-myc null phenotype in
fibroblasts with homozygous deletion of c-myc. Taken together, these data argue against the paradigm that all of the
biological functions of c-Myc are mediated by transcriptional activation of specific target genes through E box
elements (Xiao, 1998).
Cyclin E-Cdk2 kinase activation is an essential step in Myc-induced proliferation. It is presumed that this requires sequestration of G1 cell cycle inhibitors p27Kip1 and p21Cip1 (Ckis) via a Myc-induced protein. This sequestration is shown to be mediated by the protein synthesis rate of induction of cyclin D1 and/or cyclin D2. Consistent with this, primary cells from cyclin D1-/- and cyclin D2-/- mouse embryos, unlike wild-type controls, do not respond to Myc with increased proliferation, although they undergo accelerated cell death in the absence of serum. Myc sensitivity of cyclin D1-/- cells can be restored by retroviruses expressing either cyclins D1, D2 or a cyclin D1 mutant that forms kinase-defective, Cki-binding cyclin-cdk complexes. Thus, the sequestration function of D cyclins appears essential for Myc-induced cell cycle progression but dispensable for apoptosis (Perez-Roger, 1999).
The rate of the induction of cyclin D1 and/or cyclin D2 protein synthesis leads to the preferential association of p27Kip1 and p21Cip1 with cyclin D-Cdk complexes. At the same time Myc also induces cyclin E protein synthesis; the rates of induction help to promote a net gain of newly formed Cki-free cyclin E-Cdk2 complexes. These complexes become active concomitant with phosphorylation of the kinase subunit by CAK. Consistent with this model of dynamic equilibrium, cyclin E-Cdk2 kinase activity can be controlled by changes in the rates of cyclin D synthesis. Moreover, as shown with a cyclin D mutant that forms kinase-defective Cki-binding cyclin D-Cdk complexes, this link between cyclins D-Cdk and cyclin E-Cdk2 is independent of cyclin D-Cdk activity, but correlates with the ability of cyclin D-Cdk complexes to bind or sequester Ckis. This is strongly supported by the fact that the deficiency of cyclin D1-/- mouse embryo cells to respond to Myc with increased proliferation is restored by expression of the same cyclin D mutant. Consistent with these findings, transient over-expression of either catalytically inactive cyclin D-Cdk, or cyclin E-Cdk2 complexes can rescue the cell cycle inhibitory effect of a dominant-negative Mad-Myc chimera. It is concluded that due to the nature of physical interactions between cyclin D-Cdks and the cell cycle inhibitors p27Kip1 and p21Cip1, cyclin D-Cdk complexes can fulfil a dual function as cell cycle kinases and as buffers for sequestration or release of cell cycle inhibitors (Perez-Roger, 1999 and references therein).
The ability of c-MycS to repress transcription
suggests that repression of growth inhibitory genes, such as gadd45 and gas1, remains viable as an
alternative model for c-Myc molecular function. Although the transactivation-defective c-MycS protein can function in several
biological assays and can substitute for the full-length c-Myc2 in myc null cells, c-MycS may not be
able to function as full-length c-Myc2 in all assays. For example, c-MycS does not appear to cooperate
with Ras in the transformation of rat embryo fibroblasts (REFs). One explanation for these results is
that the Myc/Ras cotransformation of REF cells requires transactivation of specific myc target genes
through EMS sites that are not required for stimulation of proliferation, apoptosis, or
anchorage-independent growth. Perhaps the ability of c-Myc2 to immortalize, which may be distinct
from its ability to stimulate proliferation or induce apoptosis, is required to render REF cells susceptible
to transformation by Ras, as Ras has been shown to induce senescence.
However, one caveat in the interpretation of these negative results is that in transient transfection
assays c-MycS is expressed severalfold less in REF and other cells compared to c-Myc2. The finding of
new c-Myc target genes and perhaps new DNA-binding sites will also determine whether c-MycS has
any transactivation capabilities. Comparison of c-Myc2 and c-MycS allows the separation of the
transcriptional activation and repression abilities of c-Myc and will allow further insight into the
molecular basis for the complex and diverse biological functions of c-Myc (Xiao, 1998).
Activation of the Ras/Raf/ERK pathway extends the half-life of the Myc protein and thus enhances
the accumulation of Myc activity. Investigated were two N-terminal phosphorylation sites in
Myc, Thr 58 and Ser 62, known to be regulated by mitogen stimulation. Phosphorylation of these
two residues is critical for determining the stability of Myc. Phosphorylation of Ser 62 is required for Ras-induced stabilization of
Myc, likely mediated through the action of ERK. Conversely, phosphorylation of Thr 58, likely mediated by GSK-3 but dependent
on the prior phosphorylation of Ser 62, is associated with degradation of Myc. Further analysis demonstrates that the Ras-dependent PI-3K pathway is also
critical for controlling Myc protein accumulation, likely through the control of GSK-3 activity. These observations thus define a synergistic role for multiple
Ras-mediated phosphorylation pathways in the control of Myc protein accumulation during the initial stage of cell proliferation (Sears, 2000).
The amino acid sequence surrounding Ser 62 represents a consensus ERK recognition sequence, and evidence has been presented that ERK can
mediate the phosphorylation of Myc at Ser 62. Mutation of Ser 62 prevents mitogen- and Ras-induced stabilization of Myc. Moreover, phosphorylation at Ser 62 is enhanced under conditions
where Myc is stabilized. The importance of Ser 62 in the control of Myc stability is seen in the strict requirement
for the stabilization of Myc by Ras, but seen from work that has demonstrated an impaired transforming function when Ser 62 is altered. In contrast, phosphorylation at Thr 58 coincides with a decreased stability of Myc and mutations that prevent Thr 58 phosphorylation lead to stable Myc
protein. Once again, this coincides with work that has shown that alteration of Thr 58 enhances the transforming activity of Myc and that mutations at this site are common in Myc proteins derived from tumors. Various
lines of work suggest that the GSK-3 protein kinase is most likely responsible for the phosphorylation of Myc at Thr 58. Thr 58 lies within an established
consensus, and GSK-3 has been shown to phosphorylate Thr 58 in Myc in vitro. However,
unlike ERK, which is tightly regulated by cell growth, the level of GSK-3 protein is constant and does not fluctuate with cell growth. Nevertheless, despite the
continual presence of GSK-3 protein, the activity of the kinase is regulated during the initial phase of cell proliferation. In particular,
GSK-3 activity is inhibited through the action of PI-3K/AKT. Thus, as Ras initiates the PI-3K/AKT pathway, GSK-3 activity is held
in check, preventing the phosphorylation of Thr 58. Only when AKT activity declines would GSK-3 then have the capacity to phosphorylate Thr 58 to induce
the degradation of Myc. Thus, Ras activation elicits two responses within the cell that can cooperate to enhance Myc stability: a direct effect of ERK and an
indirect effect of AKT (Sears, 2000 and references therein).
Ski is a component of the histone deacetylase complex
required for transcriptional repression by Mad and thyroid
hormone receptor. The basic helix-loop-helix (bHLH) proteins of the Mad family act as transcriptional repressors after
heterodimerization with Max. N-CoR is required for Mad-induced transcriptional repression. The same target sequence of Mad/Max, the so-called E-box, is also recognized by a
heterodimer of Myc/Max that activates transcription. It is believed that transcriptional activation of a
group of target genes by Myc/Max enhances cellular proliferation or transformation, whereas
transcriptional repression of the same target genes by Mad/Max leads to suppression of proliferation or
induction of terminal differentiation in a wide range of cell types.
The N-CoR/SMRT complex containing mSin3 and histone deacetylase (HDAC) mediates transcriptional
repression by nuclear hormone receptors and Mad.
The oncogene v-ski was originally identified in avian Sloan-Kettering viruses, and found to transform
chicken embryo fibroblasts. Overexpression of either c-ski or v-ski induces either
transformation or muscle differentiation of quail embryo fibroblasts, depending on the growth conditions. Furthermore, v-ski transgenic mice
have increased muscle mass caused by hypertrophy of type II fast muscle fibers.
The capacity of ski to induce both transformation (growth) and differentiation, which is usually
associated with the cessation of growth, is an intriguing paradox. The human c-ski proto-oncogene
product (c-Ski) is a 728-amino-acid nuclear protein.
Recombinant c-Ski protein purified from Escherichia coli cannot directly bind to DNA, but c-Ski in
nuclear extracts from mammalian cell cultures binds to DNA, suggesting that c-Ski binds only to DNA
when associated with other proteins. The amino- and carboxy-terminal regions of
c-Ski possess a cysteine-rich and a coiled-coil region, respectively, and both regions contribute
additionally to indirect DNA binding by c-Ski. The v-Ski protein lacks 292 amino acids from the
carboxyl terminus of c-Ski, but still contains the amino-terminal cysteine-rich region. The amino-terminal region is responsible for both the cellular transformation and myogenesis
capacity of ski. The ski gene family comprises two members, ski and sno
(ski-related novel gene) and both have been shown to share clear homology in
their amino- and carboxy-terminal regions. Although it was
speculated that Ski/Sno proteins are involved in transcriptional repression of specific target genes, their function remains unknown (Nomura, 1999 and references).
The proteins encoded by the ski proto-oncogene family
directly bind to N-CoR/SMRT and mSin3A, and form a complex with HDAC. c-Ski and its related gene
product Sno are required for transcriptional repression by Mad and thyroid hormone receptor (TRbeta).
The oncogenic form, v-Ski, which lacks the mSin3A-binding domain, acts in a dominant-negative fashion,
and abrogates transcriptional repression by Mad and TRbeta. In ski-deficient mouse embryos, the ornithine
decarboxylase gene, whose expression is normally repressed by Mad-Max, is expressed ectopically. These
results show that Ski is a component of the HDAC complex and that Ski is required for the transcriptional
repression mediated by this complex. The involvement of c-Ski in the HDAC complex indicates that the
function of the HDAC complex is important for oncogenesis (Nomura, 1999).
Study of the transformation capacity of various forms of c-Ski indicate that the amino-terminal
cysteine-rich region is responsible for cellular transformation, however, the mechanism of transformation
has remained obscure. The results presented here indicate that the amino-terminal region, which is needed for cellular
transformation, is responsible for interaction with N-CoR/SMRT. Furthermore, v-Ski and the
carboxy-truncated form of c-Ski lack the carboxy-terminal mSin3A-binding domain; they abrogate
transcriptional repression by Mad by functioning in a dominant-negative fashion. Transcriptional
activation by Myc causes cell proliferation, whereas transcriptional repression by Mad inhibits cell
proliferation.
Therefore, Mad is thought to act as a tumor suppressor, and in fact, one of the mad-related genes,
mxi1, acts as a tumor suppressor using mutant mice. Therefore, abrogation of Mad-induced transcriptional repression by v-Ski may lead to induction
of Myc target genes and cellular transformation (Nomura, 1999 and references).
To address the role of N-myc in neurogenesis and in nervous system tumors, N-myc expression was conditionally disrupted in neuronal progenitor cells
(NPCs) with a nestin-Cre transgene. Null mice display ataxia, behavioral abnormalities, and tremors that correlate with a twofold
decrease in brain mass that disproportionately affects the cerebellum (sixfold reduced in mass) and the cerebral cortex, both of which
show signs of disorganization. In control mice at E12.5, a domain of high N-Myc protein expression is detected in the rapidly
proliferating cerebellar primordium. Targeted deletion of N-myc results in severely compromised proliferation as shown by a striking
decrease in S phase and mitotic cells as well as in cells expressing the Myc target gene cyclin D2, whereas apoptosis is unaffected. Null progenitor cells also have comparatively high levels of the cdk inhibitors p27Kip1 and p18Ink4c, whereas p15Ink4b, p21Cip1, and p19Ink4d levels are unaffected. Many null progenitors also exhibit altered nuclear morphology and size. In addition, loss of N-myc disrupts neuronal differentiation as evidenced by ectopic staining of the neuron specific marker ßTUBIII in the cerebrum. Furthermore, in progenitor cell cultures derived from null embryonic brain, a dramatic increase is observed in neuronal differentiation compared with controls. Thus, N-myc is essential for normal neurogenesis, regulating NPC proliferation, differentiation, and nuclear size. Its effects on proliferation and differentiation appear due, at least in part, to down-regulation of a specific subset of cyclin-dependent kinase inhibitors (Knoepfler, 2002).
Upon activation, cell surface death receptors, Fas/APO-1/CD95 and tumor necrosis factor receptor-1 (TNFR-1), are attached to cytosolic adaptor proteins, which in turn recruit caspase-8 (MACH/FLICE/Mch5) to activate the interleukin-1 beta-converting enzyme (ICE)/CED-3 family protease (caspase) cascade (see Drosophila Caspase 1). However, it remains unknown whether these apoptotic proteases are generally involved in apoptosis triggered by other stimuli, such as Myc and p53. This study suggests that a death protease cascade consisting of caspases and serine proteases plays an essential role in Myc-mediated apoptosis. When Rat-1 fibroblasts stably expressing either s-Myc or c-Myc are induced to undergo apoptosis by serum deprivation, a caspase-3 (CPP32)-like protease activity that cleaves a specific peptide substrate (Ac-DEVD-MCA) appears in the cell lysates. Induction of s-Myc- and c-Myc-mediated apoptotic cell death is effectively prevented by caspase inhibitors such as Z-Asp-CH2-DCB and Ac-DEVD-CHO. Exposing the cells to a serine protease inhibitor also significantly inhibits s-Myc- and c-Myc-mediated apoptosis and the appearance of the caspase-3-like protease activity in vivo. However, the inhibitor does not directly inhibit caspase-3-like protease activity in the apoptotic cell lysates in vitro. Together, these results indicate that caspase-3-like proteases play a critical role in both s-Myc- and c-Myc-mediated apoptosis and that caspase-3-like proteases function downstream of the protease-sensitive step in the signaling pathway of Myc-mediated apoptosis (Kagaya, 1997).
Induction of apoptosis by oncogenes like c-myc may be important in restraining the emergence of neoplasia. However, the mechanism by which c-myc induces apoptosis is unknown. CD95 (also termed Fas or APO-1) is a cell surface transmembrane receptor of the tumor necrosis factor receptor family that activates an intrinsic apoptotic suicide program in cells upon binding either its ligand CD95L or antibody. c-myc-induced apoptosis is shown to require interaction on the cell surface between CD95 and its ligand. c-Myc acts downstream of the CD95 receptor by sensitizing cells to the CD95 death signal. IGF-I signaling and Bcl-2 suppress c-myc-induced apoptosis by also acting downstream of CD95. These findings link two apoptotic pathways previously thought to be independent and establish the dependency of Myc on CD95 signaling for its killing activity (Hueber, 1997).
Pim-1 oncoprotein is a serine/threonine kinase that can closely cooperate with c-Myc in lymphomagenesis, as does Bcl-2. Although the molecular mechanism of this cooperative transformation remains unknown, it is speculated that (similar to Bcl-2) Pim-1 contributes to transformation by inhibiting apoptosis. In this study, therefore, the effect of Pim-1 expression was examined on c-Myc-mediated apoptosis of Rat-1 fibroblasts triggered by serum deprivation. Rather than inhibiting apoptosis, Pim-1 expression stimulates c-Myc-mediated apoptosis in Rat-1 fibroblasts. Pim-1 stimulates c-Myc-mediated apoptosis through an enhancement of the c-Myc-mediated activation of caspase-3 (CPP32)-like proteases, since the suppression of this activity by a specific caspase inhibitor abolishes the apoptosis stimulation by Pim-1. A kinase-defective Pim-1 mutant fails to stimulate c-Myc-mediated apoptosis; Pim-1 expression alone, in the absence of c-Myc overexpression, does not induce apoptosis of serum-deprived Rat-1 cells, indicating that the kinase activity of Pim-1 and the activated c-Myc signaling pathway are required for apoptosis stimulation by Pim-1. Together, these results suggest that Pim-1 oncoprotein stimulates as a serine/threonine kinase the death signaling elicited by c-Myc at a step upstream of caspase-3-like protease activation in Rat-1 fibroblasts. These results also suggest that Pim-1 kinase might function cooperatively with c-Myc through the phosphorylation of a factor(s) that regulates the common signaling pathway involved in c-Myc-mediated apoptosis and transformation (Mochizuki, 1997).
Nuclear factor kappaB (NF-kappaB) appears to participate in the excitotoxin-induced apoptosis of striatal medium spiny neurons. To elucidate molecular mechanisms by which this transcription factor contributes to NMDA receptor-triggered apoptotic cascades in vivo, rats were given the NMDA receptor agonist quinolinic acid (QA) by intrastriatal infusion, and the role of NF-kappaB in the induction of apoptosis-related genes and gene products was evaluated. QA administration induces time-dependent NF-kappaB nuclear translocation. The nuclear NF-kappaB protein after QA treatment is comprised mainly of p65 and c-Rel subunits as detected by gel supershift assay. Levels of c-Myc and p53 mRNA and protein are markedly increased at the time of QA-induced NF-kappaB nuclear translocation. Immunohistochemical analysis shows that c-Myc and p53 induction occurs in the excitotoxin-sensitive medium-sized striatal neurons. NF-kappaB nuclear translocation is blocked in a dose-dependent manner by the cell-permeable recombinant peptide NF-kappaB SN50, but not by the NF-kappaB SN50 control peptide. NF-kappaB SN50 significantly inhibits the QA-induced elevation in levels of c-Myc and p53 mRNA and protein. Pretreatment or posttreatment with NF-kappaB SN50, but not the control peptide, also substantially reduces the intensity of QA-induced internucleosomal DNA fragmentation. The results suggest that NF-kappaB may promote an apoptotic response in striatal medium-sized neurons to excitotoxic insult through upregulation of c-Myc and p53. This study also provides evidence indicating a unique signaling pathway from the cytoplasm to the nucleus, which regulates p53 and c-Myc levels in these neurons during apoptosis (Qin, 1999).
Human monocytic leukemia U937 cells readily undergo apoptosis when they are treated with TNF-alpha, anti-Fas antibody and anticancer drugs, such as etoposide and Ara-C. To study the mechanism of apoptosis, a novel apoptosis-resistant variant, UC, was developed from U937 cells. The UC cells show resistance to apoptosis induced by TNF-alpha, anti-Fas antibody, etoposide and Ara-C. Somatic cell hybridization between U937 and UC shows that apoptosis-resistance to TNF-alpha in UC is genetically recessive, while resistance to etoposide is dominant, suggesting that UC has at least two different mutations functionally involved in apoptosis. Mechanistic analysis reveals that UC cells express reduced amounts of c-Myc. Transfection of the c-myc gene into UC cells restores the sensitivity of the cells to undergo apoptosis induced by TNF-alpha and anti-Fas, which attributes apoptosis-resistance in this circumstance to the reduced expression of c-Myc. In contrast, c-myc transfection into UC cells can not restore their sensitivity to etoposide- and Ara-C-induced apoptosis, arguing against the role of c-myc in chemotherapy-induced apoptosis. However, treating the parental U937 cells with antisense oligonucleotides designed to reduce c-Myc expression renders the cells resistant to etoposide-induced apoptosis as well as to TNF-alpha-induced apoptosis. These results indicate that the reduced expression of c-Myc in UC is strongly associated with the resistance to etoposide-induced apoptosis. The finding that c-myc transfection into UC cannot restore the sensitivity to etoposide-induced apoptosis, suggests UC could have a second mutation that confers resistance to etoposide-induced apoptosis in a genetically dominant manner. Taken together, these present results indicate that c-Myc plays a role in cellular susceptibility to death receptor-mediated and chemotherapy-induced apoptosis (Dong, 1997).
The INK4a-ARF locus is a common target of deletion and mutation in human cancers, possibly second in frequency only to p53. The INK4a tumor suppressor locus encodes p16INK4a, an inhibitor of cyclin D-dependent kinases, and p19ARF, an alternative reading frame protein that also blocks cell proliferation. Establishment of primary mouse embryo fibroblasts (MEFs) as continuously growing cell lines is normally accompanied by loss of the p53 or p19(ARF) tumor suppressors, which act in a common biochemical pathway. Given their apparent immortalizing functions, it seems paradoxical that myc and E1A are also potent inducers of apoptosis. The sensitivity of rodent fibroblasts to myc- or E1A-induced apoptosis correlates directly with the levels of oncoprotein expression and is greatly potentiated by depriving cells of extracellular survival factors. Both Myc and E1A can induce p53 stabilization and trigger p53-dependent transcription. Several lines of evidence indicate that p53 mediates apoptosis by myc and E1A in primary fibroblasts, with p53 loss rendering cells highly resistant to their deleterious effects. For cells overexpressing myc to grow, programmed cell death must be actively suppressed. Therefore, myc overexpression should provide a strong selective pressure for events that dismantle apoptotic signaling pathways. myc rapidly activates ARF and p53 gene expression in primary MEFs and triggers replicative crisis by inducing apoptosis. MEFs that survive myc overexpression sustain p53 mutation or ARF loss during the process of establishment and become immortal. MEFs lacking ARF or p53 exhibit an attenuated apoptotic response to myc and rapidly give rise to cell lines that proliferate in chemically defined medium lacking serum. Therefore, ARF regulates a p53-dependent checkpoint that safeguards cells against hyperproliferative, oncogenic signals (Zindy, 1998).
Overexpression of the MYC protooncogene has been implicated in the genesis of diverse human tumors. Tumorigenesis induced by MYC has been
attributed to sustained effects on proliferation and differentiation. MYC may also contribute to tumorigenesis by destabilizing the cellular
genome. A transient excess of MYC activity increases tumorigenicity of Rat1A cells by at least 50-fold. The increase in tumorigenicity persists for >30 days after the return
of MYC activity to normal levels. The brief surfeit of MYC activity is accompanied by evidence of genomic instability, including karyotypic
abnormalities, gene amplification, and hypersensitivity to DNA-damaging agents. MYC also induced genomic destabilization in normal human fibroblasts,
although these cells do not become tumorigenic. Stimulation of Rat1A cells with MYC accelerates their passage through G1/S. Moreover, MYC can force
normal human fibroblasts to transit G1 and S after treatment with N-(phosphonoacetyl)-L-aspartate (PALA) at concentrations that normally lead to arrest in
S phase by checkpoint mechanisms. Instead, the cells subsequently appear to arrest in G2. It is suggest that the accelerated passage through G1 is
mutagenic but that the effect of MYC permits a checkpoint response only after G2 has been reached. Thus, MYC may contribute to tumorigenesis through
a dominant mutator effect (Felsher, 1999a).
The targeted repair of mutant protooncogenes or the inactivation of their gene products may be a specific and effective therapy for human neoplasia. To examine this
possibility, the tetracycline regulatory system has been used to generate transgenic mice that conditionally express the MYC protooncogene in hematopoietic cells.
Sustained expression of the MYC transgene culminates in the formation of malignant T cell lymphomas and acute myleoid leukemias. The subsequent inactivation of
the transgene causes regression of established tumors. Tumor regression is associated with rapid proliferative arrest, differentiation and apoptosis of tumor cells,
and resumption of normal host hematopoiesis. It is concluded that even though tumorigenesis is a multistep process, remediation of a single genetic lesion may be
sufficient to reverse malignancy (Felsher, 1999b).
The protooncogene c-myc regulates cell growth, differentiation, and apoptosis, and its aberrant expression is frequently observed in human cancer. However, the
consequences of activating c-Myc in an adult tissue, in which these cellular processes are part of normal homeostasis, remain unknown. In order to activate the protein in adult tissue, expression of a switchable form of the c-Myc protein was targeted to the skin epidermis, a well characterized homeostatic tissue. Activation of
c-MycER in adult suprabasal epidermis rapidly triggers proliferation and disrupts differentiation of postmitotic keratinocytes. Sustained activation of c-Myc is
sufficient to induce papillomatosis together with angiogenesis -- changes that resemble hyperplastic actinic keratosis, a commonly observed human precancerous
epithelial lesion. All these premalignant changes spontaneously regress upon deactivation of c-MycER (Pelengaris, 1999).
The bmi-1 and myc oncogenes collaborate strongly in murine lymphomagenesis, but previously, the basis for this collaboration has not been understood. The ink4a-ARF tumor suppressor locus is a critical downstream target of the Polycomb-group transcriptional
repressor Bmi-1. Part of Myc's ability to induce apoptosis depends on induction of p19arf. Down-regulation of ink4a-ARF by Bmi-1 underlies its ability to cooperate with Myc in tumorigenesis. Heterozygosity
for bmi-1 inhibits lymphomagenesis in Eľ-myc mice by enhancing c-Myc-induced apoptosis. Increased apoptosis is observed in bmi-1 -/- lymphoid organs. This apoptosis can be rescued by deletion of ink4a-ARF or overexpression of bcl2. Furthermore, Bmi-1 collaborates with Myc in
enhancing proliferation and transformation of primary embryo fibroblasts (MEFs) in an ink4a-ARF dependent manner, by prohibiting Myc-mediated induction of
p19arf and apoptosis. Strong collaboration is observed between the Eľ-myc transgene and heterozygosity for ink4a-ARF. This heterozygosity is accompanied by loss of the
wild-type ink4a-ARF allele and formation of highly aggressive B-cell lymphomas. Together, these results reinforce the critical role of Bmi-1 as a dose-dependent
regulator of ink4a-ARF, which in its turn acts to prevent tumorigenesis upon activation of oncogenes such as c-myc (Jacobs, 1999).
N-myc is a transcription factor expressed in the developing metanephric kidney and other organs. In mice, complete
disruption of the N-myc gene results in fetal death on the first day of renal organogenesis. In addition to the null N-myc
allele, others have generated a hypomorphic N-myc allele. In this study, combinations of these mutant genes were used to
demonstrate that reduction in N-myc protein levels correlate with fewer developing glomeruli and collecting ducts in
embryonic kidney explants. Histological sections reveal that the mutant kidneys are hypoplastic with normal
developing structures. The data indicate that the hypoplasia is due to a reduction in proliferation rather than an increase in
apoptosis. Thus, N-myc loss causes a decrease in numbers of ureteric bud tips and developing glomeruli in explants and
hypoplastic kidneys in vivo, in a dose-dependent manner (Bates, 2000).
Ids regulate differentiation1 through sequestration of basic helix-loop-helix (bHLH) transcription factors, and the consequent inhibition of their ability to bind DNA.
Although all Id proteins are viewed as positive regulators of cell-cycle progression, this role has been firmly established only for one member of the Id family, Id2. Only Id2, and not the other members of the family, Id1 and Id3, is able to disrupt the antiproliferative effects of tumor suppressor proteins of the Rb family
(the 'pocket' proteins: Rb, p107 and p130), thus allowing cell-cycle progression. This function correlates with the ability of Id2, but not Id1 and Id3, to associate
physically with active, hypophosphorylated forms of the pocket proteins in vitro and in vivo. By inactivating Rb, Id2 is also able to abolish the function of another
growth-inhibitory protein, p16, that operates upstream of Rb (Lasorella, 2000).
The Rb-null phenotype is lethal by embryonic day 14.5 because of widespread proliferation, defective differentiation and apoptosis in the nervous
system and haematopoietic precursors. Since Id2 is expressed in these cell types at the time that Rb-null embryos die1, it is hypothesized that, if Id2 is a natural target
of Rb, manifestation of the Rb-mutant phenotype might require intact Id2 (Lasorella, 2000).
Disruption of the Rb pathway (which also includes cyclin D, cdk4/6 and p16) is a hallmark of cancer and it is widely accepted that normal Rb function must be
removed, one way or another, in all human tumors. Therefore, it was of interest to determine whether tumor cells deregulate Id2 to bypass the Rb pathway. Correct
expression of Id2 is essential to regulate proliferation and differentiation of the neural crest, thus neural crest precursor cells might be sensitive to inappropriate
expression of Id2. In humans, neoplastic transformation of neural crest precursors during embryogenesis causes neuroblastoma. Interestingly, genetic
alterations of Rb, cyclin D, cdk4/6 or p16 are absent in neuroblastoma. The genetic hallmark of neuroblastoma is amplification of the gene for a member of the
Myc family of proto-oncogenes, N-myc. Resembling enforced expression of Id2, Myc overexpression is sufficient to bypass the Rb-p16 growth-inhibitory pathway, in
spite of persistent hypophosphorylated Rb. Consequently, Myc activation may release the pressure to mutate components of the Rb-p16 pathway during
tumorigenesis (Lasorella, 2000).
Id2-Rb double knockout embryos survive to term with minimal
or no defects in neurogenesis and hematopoiesis, but they die at birth from severe reduction of muscle tissue. In neuroblastoma Id2 is overexpressed in cells carrying extra copies of the N-myc gene. In these cells, Id2 is in molar excess of the active
form of Rb. The overexpression of Id2 results from transcriptional activation by oncoproteins of the Myc family. Cell-cycle progression induced by Myc
oncoproteins requires inactivation of Rb by Id2. Thus, a dual connection links Id2 and Rb: during normal cell-cycle, Rb prohibits the action of Id2 on its
natural targets, but oncogenic activation of the Myc-Id2 transcriptional pathway overrides the tumor-suppressor function of Rb (Lasorella, 2000).
Overexpression of the proto-oncogene c-myc has been implicated in the genesis of
diverse human tumors. c-Myc seems to regulate diverse biological processes, but
its role in tumorigenesis and normal physiology remains enigmatic. An allelic series of mice has been generated in which c-myc expression is
incrementally reduced to zero. Fibroblasts from these mice show reduced
proliferation and after complete loss of c-Myc function they exit the cell
cycle. Myc activity is not needed for cellular growth but does
determine the percentage of activated T cells that re-enter the cell cycle. In
vivo, reduction of c-Myc levels results in reduced body mass owing to multiorgan
hypoplasia, in contrast to Drosophila c-myc mutants, which are smaller as a
result of hypotrophy. Drosophila myc substitutes for c-myc in fibroblasts,
indicating they have similar biological activities. This suggests there may be
fundamental differences in the mechanisms by which mammals and insects control
body size. It is proposed that in mammals c-Myc controls the decision to divide or not to divide and thereby functions as a crucial mediator of signals that determine organ and body size (Trumpp, 2001).
Although the data thus appear to suggest that Myc has different
functions in Drosophila and mice, this is contradicted by evidence
that despite their relatively weak sequence homology, both c-Myc
and dMyc proteins have similar biological activities. The results
showing that dmyc expression can at least partially rescue the
proliferation defect in c-myc-deficient mouse fibroblasts
support this view and further suggest that in mouse cells dMyc can
control target genes normally regulated by c-Myc. The difference
between flies and mice may therefore lie in the identity of the target
genes that are controlled by Myc activity in each organism, or the
way in which those target genes are integrated in the genetic
circuitry of that organism. The opposite phenotypes, organ hypotrophy in Drosophila versus hypoplasia in mice, may therefore result
from differences in the way invertebrates and mammals regulate
tissue and body size. Among insect species, organ and body size
differences appear to be a function of cell number and cell size,
whereas among mammalian species they are almost exclusively due
to variations in cell number. This may be due to a tighter
coupling of cell growth and cell division in mammals than in insects. Such a link would maintain average cell size in an expanding population and would thus make tissue size determination in mammals a function of the number of cell divisions and hence a function of Myc activity (Trumpp, 2001).
Myc overexpression is a hallmark of human cancer and promotes transformation by facilitating immortalization. This function has been linked to the ability of c-Myc to induce the expression of the catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), since ectopic expression of TERT immortalizes some primary human cell types. c-Myc up-regulates telomerase activity in primary mouse embryonic fibroblasts (MEFs) and myeloid cells. Paradoxically, Myc overexpression also triggers the ARF-p53 apoptotic program, which is activated when MEFs undergo replicative crises following culture ex vivo. The rare immortal variants that arise from these cultures generally suffer mutations in p53 or delete Ink4a/ARF, and Myc greatly increases the frequency of these events. Alternative reading frame (ARF)- and p53-null MEFs have increased telomerase activity, as do variant immortal clones that bypass replicative crisis. Similarly, immortal murine NIH-3T3 fibroblasts and myeloid 32D.3 and FDC-P1.2 cells do not express ARF and have robust telomerase activity. However, Myc overexpression in these immortal cells results in remarkably discordant regulation of TERT and telomerase activity. Furthermore, in MEFs and 32D.3 cells, TERT expression and telomerase activity are regulated independently of endogenous c-Myc. Thus, the regulation of TERT and telomerase activity is complex and is also regulated by factors other than Myc, ARF, or p53 (Drissi, 2001).
To explore the role of c-Myc in carcinogenesis, a reversible transgenic model of pancreatic ß cell oncogenesis has been developed using a switchable form of the c-Myc protein. Activation of c-Myc in adult, mature ß cells induces uniform ß cell proliferation but is accompanied by overwhelming apoptosis that rapidly erodes ß cell mass. Thus, the oncogenic potential of c-Myc in ß cells is masked by apoptosis. Upon suppression of c-Myc-induced ß cell apoptosis by coexpression of Bcl-xL, c-Myc triggers rapid and uniform progression into angiogenic, invasive tumors. Subsequent c-Myc deactivation induces rapid regression associated with vascular degeneration and ß cell apoptosis. The data indicate that highly complex neoplastic lesions can be both induced and maintained in vivo by a simple combination of two interlocking molecular lesions. Recent studies with other switchable oncogene transgenic models reinforce the notion that incapacitating the driving oncogenic lesion can lead to expeditious regression of tumors induced in many different tissues by c-Myc, or even T antigens. At least in principle, therefore, the complexity of the tumor phenotype need not be instructed by an equivalent complexity of genetic or epigenetic alteration. Rather, cancers may be underpinned by only a modest number of interdependent, pleiotropic lesions that present themselves as mission-critical targets for effective cancer therapies (Pelengaris, 2002).
In most postmitotic neurons, expression or activation of proteins that stimulate cell cycle progression or DNA replication results in
apoptosis. One potential exception to this generalization is neuroblastoma (NB), a tumor derived from the sympathoadrenal
lineage. NBs often express high levels of N-myc, a proto-oncogene that can potently activate key components of the cell cycle
machinery. In postmitotic sympathetic neurons, N-myc can induce S-phase entry while protecting neurons
from death caused by aberrant cell cycle reentry. Specifically, these experiments demonstrate that expression of N-myc at levels
similar to those in NBs causes sympathetic neurons to reenter S-phase, as monitored by 5-bromo-2-deoxyuridine incorporation and expression of cell cycle
regulatory proteins, and rescues them from apoptosis induced by withdrawal of their obligate survival factor, nerve growth factor. The N-myc-induced cell cycle
entry, but not enhanced survival, is inhibited by coexpression of a constitutively hypophosphorylated form of the retinoblastoma tumor suppressor protein,
suggesting that these two effects of N-myc are mediated by separate pathways. In contrast, N-myc does not cause S-phase entry in postmitotic cortical neurons. Thus,
N-myc both selectively causes sympathetic neurons to reenter the cell cycle and protects them from apoptosis, potentially contributing to their transformation to NBs (Wartiovaara, 2002).
How does N-myc mediate this S-phase entry? The results demonstrating that coexpression of hypophosphorylated pRb rescues the BrdU incorporation argues that
N-myc mediates this effect via pRb. Such an effect could be mediated by direct interactions between N-myc and pRb, and it could also
be indirectly mediated via an N-myc-induced increase in levels of the inhibitory basic helix-loop-helix protein, Id2, which binds to hypophosphorylated pRb and
inhibits its ability to lock cells out of S-phase. An additional, potentially related mechanism involves N-myc-mediated downregulation of the cyclin-dependent kinase inhibitor p27, which in fibroblasts is essential for induction of cyclin E-cdk2 kinase activity, but not for S-phase entry. Although the data
presented here do not distinguish between these alternative explanations, it has been observed that p27 levels are decreased and Id2 levels increased in sympathetic
neurons overexpressing N-myc, suggesting that decreased p27 may collaborate with increased Id2 to trigger S-phase entry (Wartiovaara, 2002).
Results reported here also indicate that N-myc overexpression does not induce S-phase entry in cortical neurons, suggesting that sympathetic and cortical neurons
are locked out of the cell cycle via distinct mechanisms. Such a difference could be predicted by considering the development of these two populations of neurons.
Cortical neurons, like most CNS neurons, induce neuronal gene expression and undergo terminal mitosis at the same time. Perturbation
of this progenitor-to-postmitotic neuron transition, for example, via functional inhibition of the pRb family or via overexpression of Id2, leads to cellular apoptosis; in no conditions yet reported do cortical cells divide while expressing a neuronal phenotype. In contrast, sympathetic neuroblasts transition through a stage in which they express a neuronal phenotype while still dividing, suggesting that the nature of terminal mitosis differs in sympathetic versus CNS neurons. In that regard, findings
may indicate that the mechanisms locking most CNS neurons out of the cell cycle are much more stringent than for sympathetic neurons (Wartiovaara, 2002).
A somewhat surprising finding reported here is that, coincident with S-phase entry, N-myc promotes enhanced survival of sympathetic neurons in the absence of
NGF. This is particularly surprising in light of findings indicating that aberrant cell cycle entry is one of the major mechanisms whereby NGF withdrawal causes
sympathetic neuron apoptosis. In particular, NGF withdrawal causes increased cyclin D1 expression, and inhibition of cdk4 and
-6, both of which phosphorylate and activate pRb, is sufficient to delay NGF withdrawal-induced apoptosis. However, in this regard, NGF withdrawal does not cause enhanced BrdU incorporation and hypophosphorylated pRb is not, by itself, sufficient to rescue
sympathetic neurons from apoptosis. Of themselves, these findings do not necessarily argue against a role for cell cycle dysregulation in NGF withdrawal-induced
apoptosis, although they do demonstrate that this dysregulation does not actually lead to S-phase reentry. Instead, the data suggest that N-myc-induced survival
mechanisms may be 'dominant' to any apoptotic signals deriving from the coincident aberrant reentry into S-phase. Interestingly, data presented here suggest (but do
not definitively establish) that one such N-myc-mediated mechanism may involve downregulation of p75NTR (Wartiovaara, 2002).
N-myc is a true oncogene with overexpression in the sympathetic chain and adrenal medulla of transgenic mice that results, via unknown mechanisms, in malignant
neuroblastoma. The experimental and clinical data showing a strong correlation between N-myc gene amplification and poor outcome in
neuroblastoma suggest that N-myc is involved in the malignant transformation of developing sympathetic precursors or neurons, or both. On the basis of data
showing that N-myc can promote S-phase entry and survival of 'postmitotic' sympathetic neurons, a model is suggested in which N-myc contributes to malignant
neuroblastoma by either stopping sympathetic neuroblasts from exiting the cell cycle or by collaborating with other risk factors to actually transform postmitotic
neurons and cause them to reenter the cell cycle (Wartiovaara, 2002).
Overexpression of c-Myc or E2F1 sensitizes host cells to various types of apoptosis. Overexpressed c-Myc or E2F1 induces accumulation of reactive oxygen species (ROS) and thereby enhances serum-deprived apoptosis in NIH3T3 and Saos-2. During serum deprivation, MnSOD mRNA is induced by NF-kappaB in mock-transfected NIH3T3, while this induction was inhibited in NIH3T3 overexpressing c-Myc or E2F1. In these clones, E2F1 inhibits NF-kappaB activity by binding to its subunit p65 in competition with a heterodimeric partner p50. In addition to overexpressed E2F1, endogenous E2F1 released from Rb is also found to inhibit NF-kappaB activity in a cell cycle-dependent manner by using E2F1+/+ and E2F1-/- murine embryonic fibroblasts. These results indicate that E2F1 promotes apoptosis by inhibiting NF-kappaB activity (Tanaka, 2002).
Oncogene overexpression activates p53 by a mechanism posited to involve uncharacterized hyperproliferative signals. This study was carried out to determine whether such signals produce metabolic perturbations that generate DNA damage, a known p53 inducer. Biochemical, cytological, cell cycle, and global gene expression analyses revealed that brief c-Myc activation can induce DNA damage prior to S phase in normal human fibroblasts. Damage correlates with induction of reactive oxygen species (ROS) without induction of apoptosis. Deregulated c-Myc partially disables the p53-mediated DNA damage response, enabling cells with damaged genomes to enter the cycle, resulting in poor clonogenic survival. An antioxidant reduces ROS, decreases DNA damage and p53 activation, and improves survival. It is proposed that oncogene activation can induce DNA damage and override damage controls, thereby accelerating tumor progression via genetic instability (Vafa, 2002).
The cyclin-dependent kinase (CDK) inhibitors p21Cip1 and p27Kip1 are induced in response to anti-proliferative stimuli and block G1/S-phase progression through the inhibition of CDK2. Although the cyclin E-CDK2 pathway is often deregulated in tumors, the relative contribution of p21Cip1 and p27Kip1 to tumorigenesis is still unclear. The MYC transcription factor is an important regulator of the G1/S transition and its expression is frequently altered in tumors. It has been suggested that p27Kip1 is a crucial G1 target of MYC. In mice, deficiency for p27Kip1 but not p21Cip1 results in decreased survival to retrovirally-induced lymphomagenesis. Importantly, in such p27Kip1 deficient lymphomas an increased frequency of Myc activation is observed. p27Kip1 deficiency also collaborates with MYC overexpression in transgenic lymphoma models. Thus, in vivo, the capacity of MYC to promote tumor growth is fully retained and even enhanced upon p27Kip1 loss. In lymphocytes, MYC overexpression and p27Kip1 deficiency independently stimulate CDK2 activity and augment the fraction of cells in S phase, in support of their distinct roles in tumorigenesis (Martins, 2002).
Myc and E2f1 promote cell cycle progression, but overexpression of either can trigger p53-dependent apoptosis. Mice expressing an Eμ-Myc transgene in B lymphocytes develop lymphomas, the majority of which sustain mutations of either Arf (a tumor suppressor whose product inhibits Mdm2, thereby stabilizing p53) or p53. Eμ-Myc transgenic mice lacking one or both E2f1 alleles exhibit a slower onset of lymphoma development associated with increased expression of the cyclin-dependent kinase inhibitor p27Kip1 and a reduced S phase fraction in precancerous B cells. In contrast, Myc-induced apoptosis and the frequency of Arf and p53 mutations in lymphomas were unaffected by E2f1 loss. Therefore, Myc does not require E2f1 to induce Arf, p53, or apoptosis in B cells, but depends upon E2f1 to accelerate cell cycle progression and downregulate p27Kip1 (Baudino, 2003).
The MYC oncoprotein is a transcription factor that coordinates cell growth and division. MYC overexpression exacerbates genomic instability and sensitizes cells to apoptotic stimuli. MYC directly stimulates transcription of the human Werner syndrome gene, WRN, which encodes a conserved RecQ helicase. Loss-of-function mutations in WRN lead to genomic instability, an elevated cancer risk, and premature cellular senescence. The overexpression of MYC in WRN syndrome fibroblasts or after WRN depletion from control fibroblasts leads to rapid cellular senescence that can not be suppressed by hTERT expression. It is proposed that WRN up-regulation by MYC may promote MYC-driven tumorigenesis by preventing cellular senescence (Grandori, 2003).
Alterations in c-myc oncogene expression have been implicated in the pathogenesis of several human cancers, including Burkitt and diffuse large B-cell lymphomas, breast and prostate cancer, colon cancer, melanoma, and multiple myeloma. The proteins encoded by MYC transcriptional target genes appear to regulate cell-cycle progression and cell growth while sensitizing cells to apoptotic stimuli. MYC may also be able to promote tumorigenesis by up-regulating the expression of genes such as hTERT that play a role in cellular immortalization or the escape from senescence. It was reasoned that MYC might modulate the expression of other genes that control cellular senescence, and thus determined whether the gene encoding the Werner syndrome RecQ helicase protein is a MYC transcriptional target (Grandori, 2003).
Werner syndrome (WRN) is an uncommon, autosomal recessive genetic instability syndrome that results from loss-of-function mutations in the chromosome 8p12-p11.2 WRN gene. The WRN phenotype resembles premature aging, and includes genomic instability, an elevated risk of malignancy, and accelerated cellular senescence. Genetic instability following loss of the 162-kD WRN RecQ helicase protein reflects the physiologic role of WRN in mitotic recombination and repair. Conversely, the elevated levels of WRN observed in immortalized and human tumor cell lines may help insure continuous cell proliferation. In order to delineate potential interactions between MYC and WRN in tumorigenesis, whether WRN expression is modulated by MYC was determined, and cellular responses to MYC overexpression in the absence of WRN were monitored. The results indicate that WRN expression appears to be required to avoid cellular senescence upon MYC up-regulation in hTERT-immortalized fibroblasts (Grandori, 2003).
Mnt is a Max-interacting transcriptional repressor that has been hypothesized to function as a Myc antagonist. To investigate Mnt function the Mnt gene was deleted in mice. Since mice lacking Mnt are born severely runted and typically die within several days of birth, mouse embryo fibroblasts (MEFs) derived from these mice and conditional Mnt knockout mice were used in this study. In the absence of Mnt, MEFs prematurely enter the S phase of the cell cycle and proliferated more rapidly than Mnt+/+ MEFs. Defective cell cycle control in the absence of Mnt is linked to upregulation of Cdk4 and cyclin E and the Cdk4 gene appears to be a direct target of Mnt-Myc antagonism. Like MEFs that overexpress Myc, Mnt-/- MEFs are prone to apoptosis, efficiently escape senescence and can be transformed with oncogenic Ras alone. Consistent with Mnt functioning as a tumor suppressor, conditional inactivation of Mnt in breast epithelium leads to adenocarinomas. These results demonstrate a unique negative regulatory role for Mnt in governing key Myc functions associated with cell proliferation and tumorigenesis (Hurlin, 2003).
Epidemiological findings suggest that the consequences of a given oncogenic stimulus vary depending upon the developmental state of the target tissue at the time of exposure. This is particularly evident in the mammary gland, where both age at exposure to a carcinogenic stimulus and the timing of a first full-term pregnancy can markedly alter the risk of developing breast cancer. Analogous to this, the biological consequences of activating oncogenes, such as MYC, can be influenced by cellular context both in terms of cell lineage and cellular environment. In light of this, it was hypothesized that the consequences of aberrant MYC activation in the mammary gland might be determined by the developmental state of the gland at the time of MYC exposure. To test this hypothesis directly, a doxycycline-inducible transgenic mouse model was used to overexpress MYC during different stages of mammary gland development. Using this model, it was found that the ability of MYC to inhibit postpartum lactation is due entirely to its activation within a specific 72-hour window during mid-pregnancy; by contrast, MYC activation either prior to or following this 72-hour window has little or no effect on postpartum lactation. Surprisingly, it was found that MYC does not block postpartum lactation by inhibiting mammary epithelial differentiation, but rather by promoting differentiation and precocious lactation during pregnancy, which in turn leads to premature involution of the gland. This developmental stage-specific ability of MYC to promote mammary epithelial differentiation is tightly linked to its ability to downregulate caveolin 1 and activate Stat5 in a developmental stage-specific manner. These findings provide unique in vivo molecular evidence for developmental stage-specific effects of oncogene activation, as well as the first evidence linking MYC with activation of the Jak2-Stat5 signaling pathway (Blakely, 2005).
ß-catenin signaling is heavily involved in organogenesis. This study
investigated how pancreas differentiation, growth and homeostasis are affected
following inactivation of an endogenous inhibitor of ß-catenin,
adenomatous polyposis coli (Apc). In adult mice, Apc-deficient pancreata are
enlarged, solely as a result of hyperplasia of acinar cells, which accumulate
ß-catenin, with the sparing of islets. Expression of a target of
ß-catenin, the proto-oncogene c-myc (Myc), is
increased in acinar cells lacking Apc, suggesting that c-myc
expression is essential for hyperplasia. In support of this hypothesis, it was
found that conditional inactivation of c-myc in pancreata lacking Apc
completely reverse the acinar hyperplasia. Apc loss in organs such as the
liver, colon and kidney, as well as experimental misexpression of c-myc in pancreatic acinar cells, lead to tumor formation with high penetrance. Surprisingly, pancreas tumors failed to develop following conditional pancreas Apc inactivation. In Apc-deficient acini of aged mice, these studies revealed a cessation of their exaggerated proliferation and a reduced expression of c-myc, in spite of the persistent accumulation of ß-catenin. In conclusion, this work shows that ß-catenin
modulation of c-myc is an essential regulator of acinar growth
control, and unveils an unprecedented example of Apc requirement in the pancreas that is both temporally restricted and cell-specific. This provides new insights into the mechanisms of tumor pathogenesis and tumor suppression in the pancreas (Strom, 2007).
Inhibition of protein phosphatase 2A (PP2A) activity has been identified as a prerequisite for the transformation of human cells. However, the molecular mechanisms by which PP2A activity is inhibited in human cancers are currently unclear. In this study, a cellular inhibitor of PP2A with oncogenic activity is described. The protein, designated Cancerous Inhibitor of PP2A (CIP2A), interacts directly with the oncogenic transcription factor c-Myc, inhibits PP2A activity toward c-Myc serine 62 (S62), and thereby prevents c-Myc proteolytic degradation. In addition to its function in c-Myc stabilization, CIP2A promotes anchorage-independent cell growth and in vivo tumor formation. The oncogenic activity of CIP2A is demonstrated by transformation of human cells by overexpression of CIP2A. Importantly, CIP2A is overexpressed in two common human malignancies, head and neck squamous cell carcinoma (HNSCC) and colon cancer. Thus, these data show that CIP2A is a human oncoprotein that inhibits PP2A and stabilizes c-Myc in human malignancies (Junttila, 2007).
FoxO transcription factors play critical roles in cell cycle control and cellular stress responses, and abrogation of FoxO function promotes focus formation by Myc in vitro. Stable introduction of a dominant-negative FoxO moiety (dnFoxO) into Eµ-myc transgenic hematopoietic stem cells accelerates lymphoma development in recipient mice by attenuating Myc-induced apoptosis. When expressed in Eµ-myc; p53+/- progenitor cells, dnFoxO alleviates the pressure to inactivate the remaining p53 allele in upcoming lymphomas. Expression of the p53 upstream regulator p19Arf (alternative reading frame of p16INK, also called p14arf in humans and p19arf in mice) is virtually undetectable in most dnFoxO-positive Myc-driven lymphomas. It was found that FoxO proteins bind to a distinct site within the Ink4a/Arf locus and activate Arf expression. Moreover, constitutive Myc signaling induces a marked increase in nuclear FoxO levels and stimulates binding of FoxO proteins to the Arf locus. These data demonstrate that FoxO factors mediate Myc-induced Arf expression and provide direct genetic evidence for their tumor-suppressive capacity (Bouchard, 2007).
The FoxO subclass of forkhead-box transcription factors (consisting of FoxO1 (FKHR), FoxO3a (FKHRL1), FoxO4 (AFX), and FoxO6) regulates numerous cellular functions including proliferation, stress sensitivity, and survival; it has also been implicated in the regulation of organism life span. The members of this family activate gene expression via interaction with a specific DNA sequence, and known targets include the cell cycle regulating Kip1, the proapoptotic Bim, the DNA damage-responsive Gadd45a, and the oxidative stress-protective manganese superoxide dismutase genes. In addition, FoxO proteins can repress several cell cycle promoting genes (e.g., cyclin D1 and cyclin D2) in a manner that might be independent of direct DNA binding (Bouchard, 2007 and references therein).
In response to growth factor signaling and to oxidative stress, FoxO proteins are post-translationally modified by phosphorylation, acetylation, and ubiquitination; collectively, these modifications regulate FoxOs’ subcellular localization, transcriptional activity, and stability. Notably, all FoxO proteins are inhibited by protein kinase B/Akt-mediated phosphorylation that promotes their nuclear export and subsequent proteolytic degradation via ubiquitination by the SCFSkp2 complex. As a consequence, FoxO proteins mediate the induction of p27Kip1 and Bim expression in response to inhibition of the phosphatidylinositol-3-OH (PI3)-kinase/Akt pathway (Bouchard, 2007 and references therein).
Conditional codeletion of the FoxO1, FoxO3, and FoxO4 alleles uncovers a context-dependent cancer-prone phenotype characterized by thymic lymphomas forming in some and hemangiomas developing in most animals after a long latency, suggesting that FoxO proteins exert their tumor-suppressive capability in the presence of additional oncogenic mutations. In support of this view, Akt-mediated phosphorylation of FoxO proteins has been identified as the critical PI3-kinase signaling component that substitutes for oncogenic Ras in Myc-induced proliferation and focus formation in vitro. Furthermore, constitutive Akt signaling cooperates with Myc to accelerate B-cell lymphomagenesis; however, it remains unclear whether Akt-mediated phosphorylation of FoxO proteins contributes to Eµ-myc transgenic lymphoma formation in this setting (Bouchard, 2007).
Proapoptotic Arf/p53 signaling is known as the pivotal Myc-induced tumor-suppressive barrier. Eµ-myc transgenic mice lacking one p53 allele develop lymphomas that inactivate the remaining wild-type allele. Likewise, Eµ-myc; Arf+/- or Eµ-myc; Ink4a/Arf+/- mice produce tumors that lack expression of p19Arf. Primary Arf deletions protect cells from acquiring p53 mutations during lymphoma development. Similarly, introduction of strictly anti-apoptotic genes such as bcl2 or a dominant-negative form of caspase 9 into Eµ-myc; p53+/- hematopoietic stem cells alleviates the pressure to inactivate p53, thereby underscoring apoptosis as the critical p53-governed tumor suppressor function in Myc-driven lymphomagenesis (Bouchard, 2007).
Previous work has shown that p53 and FoxO3a share target genes and that FoxO3a can activate transcription via p53 sites, suggesting a potential collaboration of FoxO3a and p53 in tumor suppression. Although a direct interaction between FoxO3a and p53 proteins has been demonstrated under conditions of overexpression, the observed collaboration would be consistent with an as-yet-unidentified FoxO target acting upstream of p53. This study reports that FoxO factors elicit their tumor-suppressive potential as critical inducers of Arf during Myc-driven lymphomagenesis, providing further evidence for a close link between the FoxO and p53 tumor suppressor pathways (Bouchard, 2007).
Studying the early stages of cancer can provide important insight into the molecular basis of the disease. A preneoplastic stage was identified in the patched (ptc) mutant mouse, a model for the brain tumor medulloblastoma. Preneoplastic cells (PNCs) are found in most ptc mutants during early adulthood, but only 15% of these animals develop tumors. Although PNCs are found in mice that develop tumors, the ability of PNCs to give rise to tumors has never been demonstrated directly, and the fate of cells that do not form tumors remains unknown. Using genetic fate mapping and orthotopic transplantation, definitive evidence was provide that PNCs give rise to tumors, and the predominant fate of PNCs that do not form tumors is differentiation. Moreover, N-myc, a gene commonly amplified in medulloblastoma, can dramatically alter the fate of PNCs, preventing differentiation and driving progression to tumors. Importantly, N-myc allows PNCs to grow independently of hedgehog signaling, making the resulting tumors resistant to hedgehog antagonists. These studies provide the first direct evidence that PNCs can give rise to tumors, and demonstrate that identification of genetic changes that promote tumor progression is critical for designing effective therapies for cancer (Kessler, 2009).
The Myc protein suppresses the transcription of several cyclin-dependent kinase inhibitors (CKIs) via binding to POZ domain/zinc finger transciption factor Miz1.
Whether this interaction is important for Myc's ability to induce or maintain tumorigenesis is not known. This study shows that the oncogenic potential of a point mutant of Myc (MycV394D) that is selectively deficient in binding to Miz1 is greatly attenuated. Binding of Myc to Miz1 is continuously required to repress CKI expression and inhibit accumulation of trimethylated histone H3 at Lys 9 (H3K9triMe), a hallmark of cellular senescence, in T-cell lymphomas. Lymphomas that arise express high amounts of transforming growth factor beta-2 (TGFbeta-2) and TGFbeta-3. Upon Myc suppression, TGFbeta signaling is required to induce CKI expression and cellular senescence and suppress tumor recurrence. Binding of Myc to Miz1 is required to antagonize growth suppression and induction of senescence by TGFbeta. Since lymphomas express high levels of TGFbeta, they are poised to elicit an autocrine program of senescence upon Myc inactivation, demonstrating that TGFbeta is a key factor that establishes oncogene addiction of T-cell lymphomas (van Riggelen, 2010).
The ubiquitous deregulation of Myc in human cancers makes it an intriguing therapeutic target, a notion supported by recent studies in Ras-driven lung tumors showing that inhibiting endogenous Myc triggers ubiquitous tumor regression. However, neither the therapeutic mechanism nor the applicability of Myc inhibition to other tumor types driven by other oncogenic mechanisms is established. This study shows that inhibition of endogenous Myc also triggers ubiquitous regression of tumors in a simian virus 40 (SV40)-driven pancreatic islet tumor model. Such regression is presaged by collapse of the tumor microenvironment and involution of tumor vasculature. Hence, in addition to its diverse intracellular roles, endogenous Myc serves an essential and nonredundant role in coupling diverse intracellular oncogenic pathways to the tumor microenvironment, further bolstering its credentials as a pharmacological target.
diminutive:
Biological Overview
| Regulation
| Developmental Biology
| Effects of Mutation
| References
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