Retinoblastoma-family protein
Retinoblastoma proteins, cell cycle arrest, and apoptosis E2F1 overexpression has been shown to induce apoptosis in cooperation with p53. Using Saos-2 cells, which are null for p53 and lack functional Rb, it has been shown that E2F1 overexpression can also induce apoptosis in the absence of p53 and retinoblastoma protein (Rb). E2F1-induced apoptosis can be specifically inhibited by Rb but not mdm2, which is known for its ability to inhibit p53-induced apoptosis. Through the study of the apoptotic function of a set of E2F1 mutants, it has become clear that the transactivation and the apoptotic function of E2F1 are uncoupled. Transactivation-defective E2F1 mutants can induce apoptosis as effectively as wild-type E2F1. In contrast to E2F1 transactivation, the DNA-binding activity of E2F1 is essential for its apoptotic function, as the DNA-binding-defective mutants failed to induce apoptosis. Therefore Rb may inhibit E2F1-induced apoptosis by mechanisms other than the suppression of the transactivation of E2F1. This hypothesis was supported by the observation that although Rb overexpression can specifically repress the apoptosis induced by wild-type E2F1 and an Rb-binding-competent E2F1 mutant, such overexpression fails to inhibit the apoptosis induced by mutants that are defective or reduced in Rb binding and transactivation. All of these points argue for a novel function for E2F1 and Rb in controlling apoptosis. The results also indicate that transcriptional repression rather than the transactivation function of E2F1 may be involved in its apoptotic function. It is thought that the Rb-E2F1 complex actively represses genes involved in apoptosis (Hsieh, 1997).
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. 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).
To maintain genome stability, cells with damaged DNA must arrest to allow repair of mutations before replication. Although
several key components required to elicit this arrest have been discovered, much of the pathway remains elusive. pRB is shown to act as a central mediator of the proliferative block induced by a diverse range of DNA damaging stimuli.
Rb-/- mouse embryo fibroblasts are defective in arrest after gamma-irradiation, UV irradiation, and treatment with a variety
of chemotherapeutic drugs. In contrast, the pRB related proteins p107 and p130 do not play an essential part in the DNA
damage response. pRB is required specifically for the G1/S phase checkpoint induced by gamma-irradiation. Despite a
defect in G1/S phase arrest, levels of p53 and p21 are increased normally in Rb-/- cells in response to gamma-irradiation.
These results have led to the proposal of a model in which pRB acts as an essential downstream target of the DNA damage-induced
arrest pathway. To play a part in irradiation-induced arrest, pRB must respond to upstream signals in the DNA damage
pathway. p53 is a required component of this signaling pathway. Its ability to elicit G1/S phase arrest
after irradiation is thought to be due, at least in part, to transcriptional activation of p21. DNA
damage-induced arrest is presumed to be enforced by the ability of p21 to inhibit phosphorylation of one
or more critical CDK substrates, but the identity of targets involved in the irradiation response has so
far remained elusive. It is shown here that in Rb-/- cells, p53 and p21 levels are increased normally in
response to gamma-irradiation, yet the G1/S phase arrest is defective. It is therefore suggested
that pRB acts as a central downstream target of the p53-mediated arrest pathway. It is most likely that
up-regulation of p21 acts to prevent CDK phosphorylation of pRB and therefore maintains pRB in the
growth suppressive state. UV irradiation engages a checkpoint pathway
that inactivates CDK4 via tyrosine phosphorylation. It is likely that this serves to inhibit
CDK4-mediated phosphorylation of pRB and therefore maintains pRB in an active, growth inhibitory
state (Harrington, 1998).
How does modulation of pRB activity by DNA damage signaling pathways lead to G1/S phase arrest?
While pRB has been reported to bind to a multitude of cellular proteins, its actions in cell cycle
progression are primarily attributed to binding of the E2F family of transcription factors. Binding of
pRB to the E2Fs is thought to repress the transactivation of a set of genes required for cell cycle
progression. Thus, although it is conceivable that pRB mediates DNA damage-induced arrest via
modulation of other associated proteins, it seems most likely that its actions are primarily due to altered
regulation of E2F target genes. With a few notable exceptions, it is not yet possible to
determine which E2F target genes are regulated by pRB in vivo.
The ability of pRB to prevent replication of damaged DNA is likely to inhibit the propagation of
carcinogenic mutations and may therefore contribute to its role as a tumor suppressor. Furthermore, because many cancer
therapies act by damaging DNA, these findings also have implications for the treatment of tumors in which pRB is
inactivated (Harrington, 1998).
The mammalian SWI-SNF complex is an evolutionarily conserved, multi-subunit machine, involved in
chromatin remodelling during transcriptional activation. Within this complex, the BRM (SNF2alpha) and
BRG1 (SNF2beta) proteins are mutually exclusive subunits, which are believed to affect nucleosomal
structures using the energy of ATP hydrolysis. mBRG1 protein is expressed at high levels in both embryonic and
extraembryonic tissues (yolk sack and alantoid) from embryos of 7.5, 9, 12, 15 and 18 days post-coitum. In contrast, mBRM is present at very low levels at all stages of development in the
embryonic tissue (20- to 30-fold less than mBRG1). mBRM expression was more elevated in the
extraembryonic tissue, but still lower than mBRG1 levels. This situation changes after birth; the levels of mBRM protein surpass those of mBRG1 in some organs of the adult mouse.
In order to characterize possible differences in the
function of BRM and BRG1, and to gain further insight into the role of BRM-containing SWI-SNF
complexes, the mouse BRM gene was inactivated by homologous recombination. BRM-/- mice develop
normally, suggesting that an observed up-regulation of the BRG1 protein can functionally replace BRM
in the SWI-SNF complexes of mutant cells. mBRG1 protein levels in mBRM-/- brain, liver, spleen
and kidneys are higher than those of the equivalent organs in wild-type mice. Strikingly, the increase in mBRG1 levels
is more pronounced in organs that contain high levels of mBRM in the wild-type animals (~5- to 6-fold
increase in brain, as compared with a 2-fold increase in liver and spleen). In contrast, no changes are
observed in the protein levels of other subunits of the complex such as SNF5 and BAF155. Immunoprecipitaiton experiments show that mBRG1 can
replace mBRM in the fraction of SWI-SNF complexes that mBRM usually occupies.
Adult mutant mice are ~15% heavier than
control littermates. This may be caused by increased cell proliferation, as demonstrated by a higher
mitotic index detected in mutant livers. This is supported further by the observation that mutant
embryonic fibroblasts are significantly deficient in their ability to arrest in the G0/G1 phase of the cell
cycle in response to cell confluency or DNA damage. These studies suggest that BRM participates in
the regulation of cell proliferation in adult mice (Reyes, 1998).
The Drosophila brahma gene is strongly expressed throughout embryogenesis and in pupae, but much
lower amounts are present in larvae and adult flies. This is reminiscent of the expression pattern of mBRG1. The kinetics of mBRM expression
seem to be the opposite: low during embryonic development and higher in adult tissues. This may
explain why no alterations in the developmental program are observed in the mBRM-/- animals,
especially homeotic transformations, which have been found in Drosophila brahma mutants. These data suggest that BRG1 may have a role similar to that of brahma during
development. In fact, it has been shown that no viable embryonic carcinoma F9 cells lacking
both copies of mBRG1 can be obtained, suggesting that during early
development, when mBRM is absent, mBRG1 is an essential gene. Several observations suggest that mBRM accumulates in slowly growing or G0-arrested cells. (1) In
comparison with mBRG1, mBRM expression levels throughout development, when rapid cell division
occurs, are rather low. While BRG1 expression is constitutive, zygotic expression of mBRM
begins at the blastocyst stage, when the first differentiation occurs in the embryonic tissues. In addition, mBRM is not expressed in ES cells or in F9 teratocarcinoma cells, which
display a very short G1 phase. mBRM expression is induced in these cells upon differentiation with
retinoic acid or in embryonic bodies. (2) In adult mice, mBRM is strongly expressed in post-mitotic cell types, such
as neurons. (3) Serum-deprived or contact-inhibited cells from
different origins (MEFs, NIH 3T3, HeLa, mouse mammary gland epithelial cells, HC11) contain 3- to
10-fold more BRM protein than exponentially growing cells. Under these conditions, BRG1 levels either remain constant or decrease. (4) BRM
has been found to be down-regulated in several transformed cell lines.
All of these data suggest a differential role for BRM and BRG1 in the control of genes required for
quiescence or terminal differentiation. Still other experiments suggest that both proteins share similar
properties. Both hBRM and hBRG1 have been shown to interact with members of the pRb family. This
interaction has been mapped to an LXCXE sequence present in the C-terminal region of both proteins.
Furthermore, both hBRM and hBRG1 are able to induce growth arrest of SW13 cells, which have
wild-type pRb but no detectable levels of hBRM and hBRG1. These data suggest that both hBRM and hBRG1 may cooperate in pRb-dependent
regulation of gene expression, BRM being used preferentially in G0-arrested cells. It has recently been shown
that hBRM cooperates in Rb-E2F-mediated repression of gene expression in transient
transfection studies. Consistent with this observation, it has been found that
disruption of mBRM affects the balance between proliferating and non-proliferating cells in the animal.
In fact, there is an increase in the fraction of S phase cells in mBRM-/- livers. Also, confluent or UV-irradiated mBRM-/- mouse embryonic fibroblasts (MEFs) are able to partially overcome G0/G1 checkpoints.
Furthermore, the increase in proliferation of mBRM-/- MEFs upon DNA damage correlates with an
increase in the percentage of apoptotic cells, suggesting that cells that overcome G1 arrest undergo
apoptosis. Inappropriate override of G1 arrest after DNA damage or serum deprivation also leads to
apoptosis in Rb-/- MEFs or in E2F-overexpressing cells, reinforcing a connection between the pRb pathway of regulation of
G1/S transition and the mBRM-containing SWI-SNF complexes. How could the mBRM-associated SWI-SNF complexes suppress proliferation? An obvious possibility,
deduced from the data discussed above, is that mBRM-associated SWI-SNF complexes may assist the
pRb-E2F complex in repressing genes essential for S phase entry. This is a slightly unorthodox
suggestion since the SWI-SNF complex has been considered until now as a transcriptional activator. However, SWI-SNF-induced accessibility of nucleosomal DNA
may promote events other than transcriptional activation, and it is likely that chromatin remodelling
activities are also required for binding of transcriptional repressors such as pRb-E2F complexes. It has also been shown recently that histone deacetylation is involved in pRb-dependent repression. It is possible that the BRM-containing SWI-SNF complexes might facilitate histone
deacetylation by loosening the nucleosomal structure. However, the possibility that SWI-SNF complexes may be required to activate transcription of G0- or quiescence-specific
genes cannot be formally excluded. These two possibilities are not mutually exclusive (Reyes, 1998 and references).
Mice mutant for the Rb tumor suppressor gene die in mid-gestation with defects in erythropoiesis, cell cycle control, and apoptosis.
Embryos mutant for both Rb and its downstream target E2f-1 demonstrate significant suppression of apoptosis and S phase entry in certain tissues compared to Rb mutants, implicating E2f-1 as a critical mediator of these effects. Up-regulation of the
p53 pathway, required for cell death in these cells in Rb mutants, is also suppressed in the Rb/E2f-1 double mutants. However, double
mutants have defects in cell cycle regulation and apoptosis in some tissues and die at approximately E17.0 with anemia and defective
skeletal muscle and lung development, demonstrating that E2F-1 regulation is not the sole function of pRB in development (Tsai, 1999).
Mice lacking functional Rb die at midgestation and
exhibit ectopic DNA synthesis, apoptosis, and incomplete
differentiation during neurogenesis, erythropoiesis, and
lens development. Temporal and genetic analyses are described for partially rescued Rb mutant fetuses, mgRb:Rb-/-, that survive to birth. Specific defects in skeletal muscle differentiation are revealed. Increased apoptosis, bona fide endoreduplication, and incomplete differentiation throughout terminal myogenesis exhibited by mgRb:Rb-/- fetuses is further augmented in composite mutant fetuses, mgRb:Rb-/-:p21-/-, lacking both Rb and the cyclin-dependent kinase inhibitor p21Waf1/Cip1. Although E2F1 and p53 mediate ectopic DNA synthesis and cell death in several tissues in Rb mutant embryos, both endoreduplication and apoptosis persist in mgRb:Rb-/-:E2F1-/- and mgRb:Rb-/-:p53-/- compound mutant muscles. Thus, combined inactivation of Rb and p21Waf1/Cip1 augments endoreduplication and apoptosis, whereas
E2F1 and p53 are dispensable during aberrant myogenesis in Rb-deficient fetuses.
These results show that ectopic DNA synthesis and apoptosis in
Rb-deficient muscles are mediated by a pathway, yet to be defined, which is independent of E2F1 or p53. Inactivation of both Rb and p21Waf1/Cip1 leads to increased endoreduplication and apoptosis, indicating that these two negative regulators cooperate to facilitate cell cycle exit during terminal myogenesis (Jiang, 2000).
Smad3 is a direct mediator of transcriptional activation by the TGFß receptor. Its target genes in epithelial cells include cyclin-dependent kinase inhibitors that generate a cytostatic reponse. This study defines how, in
the same context, Smad3 can also mediate transcriptional repression of the growth-promoting gene c-myc. A
complex containing Smad3, the transcription factors E2F4/5 and DP1, and the corepressor p107 preexists in the cytoplasm. In response to TGFbeta, this complex moves into the nucleus and associates with Smad4, recognizing a composite Smad-E2F site on c-myc for repression. Previously known as the ultimate recipients of cdk regulatory signals, E2F4/5 and p107 act here as transducers of TGFbeta receptor signals upstream of cdk. Smad proteins therefore mediate transcriptional activation or repression depending on their associated partners (Chen, 2002).
E2F-mediated gene repression plays a key role in regulation of neuron survival
and death. However, the key molecules involved in such regulation and the
mechanisms by which they respond to apoptotic stimuli are largely unknown.
This study shows that p130 is
the predominant Rb family member associated with E2F in
neurons, that its major partner for repression of pro-apoptotic genes is E2F4,
and that the p130-E2F4 complex recruits the chromatin modifiers HDAC1 and
Suv39H1 to promote gene silencing and neuron survival. Apoptotic stimuli induce
neuron death by sequentially causing p130 hyperphosphorylation, dissociation of
p130-E2F4-Suv39H1-HDAC complexes, altered modification of H3 histone and gene
derepression. Experimental suppression of such events blocks neuron death while
interference with the synthesis of E2F4 or p130, or with the interaction of
E2F4-p130 with chromatin modifiers, induces neuron death. Thus, neuron survival
and death are dependent on the integrity of E2F4-p130-HDAC/Suv39H1 complexes (Liu, 2005).
Active repression of E2F-regulated genes by Rb family members is achieved by
recruitment of chromatin-modifying proteins to complexes with E2F.
Thus, a key finding here is that the mechanism by which
p130-E2F4 complexes promote neuron survival is via gene repression that requires
their interaction with the chromatin-modifying corepressors HDAC and Suv39H1.
Moreover, apoptotic stimuli induce death by causing loss of such
interactions. In support of this, p130 and p130-E2F4 fusion proteins mutated to
abolish interaction with HDAC and Suv39H1 promote neuron death, while
phosphorylation-resistant E2F4-p130 fusion proteins that do not lose association
with HDAC or Suv39H1 under apoptotic conditions are protective. Although these
studies identified HDAC1 as a partner for p130 in neuronal cells, preliminary
findings indicate that other HDAC family members may also be involved in
p130-mediated gene repression and neuron survival (Liu, 2005).
How might p130-tethered HDAC1 and Suv39H1 promote gene repression and neuron
survival? One target for these enzymes is the N-terminal tail of histone H3. In
the absence of corepressors, H3 is phosphorylated on Ser 10, and this
facilitates acetylation of Lys 14. These modifications promote transcription and
are essential for cell cycle progression in mitotically competent cells.
When tethered to chromatin by Rb-E2F complexes, Suv39H1
methylates H3 residue Lys 9. This, in
turn, inhibits phosphorylation of Ser 10 and, in concert with HDACs, favors
deacetylation of Lys 14. Such changes lead to condensation of local chromatin
and gene silencing. Consistent with this mechanism, it was
observed that levels of Ac-p-H3 associated with the endogenous B-myb promoter
are low in viable neuronal cells and greatly increase in response to an
apoptotic stimulus. In support of the involvement of HDAC and Suv39H1, it was found
that a death stimulus abolishes the association between p130 and HDAC and
substantially diminishes levels of p130-associated HMT activity (Liu, 2005).
An issue raised by these studies is the target of E2F-mediated gene repression
that regulates neuron survival and death. A variety of observations support the
closely related B-myb and C-myb genes in such a role. The promoters for these
genes contain E2F-binding sites, and their expression is subject to
E2F-dependent repression. Apoptotic stimuli, including p130
down-regulation, induce Myb reporter activity in neuronal cells, and the
findings in this study show that apoptotic stimuli lead to loss of repressive complexes
containing E2F4-p130-HDAC-Suv39H1 from the endogenous B-myb promoter as well as
changes in associated chromatin consistent with gene derepression. Moreover,
B-myb and C-myb transcripts and proteins are significantly induced by apoptotic
stimuli, and overexpression of B-myb and C-myb promotes neuron death.
Finally, down-regulation of B-myb and C-myb with anti-sense and siRNA
constructs protects neurons from apoptotic stimuli (Liu, 2005).
These findings strongly support a repression/derepression model for regulation of
neuron survival/death by E2F4-p130 and associated chromatin modifiers. They
identify both the molecules and mechanisms by which p130 promotes silencing of
E2F-responsive genes in viable neurons and by which apoptotic stimuli lead to
derepression of E2F-responsive genes and death (Liu, 2005).
p130 has been implicated as a promoter of quiescence in nonneuronal cells and
may well contribute to the post-mitotic state of neurons. Thus, stimuli that
lead to dissolution of p130 complexes and that thereby trigger the derepression
of pro-apoptotic genes such as Mybs, might concomitantly stimulate neurons to
attempt cell cycle re-entry. Such a situation may explain not only why a variety
of cell cycle markers are observed in neurons affected by injury and
neurodegenerative disorders, but also why treatments targeted at suppressing
derepression of E2F-regulated genes in neurons may have a therapeutic benefit in
preventing neuron degeneration. In this regard, the present findings provide several additional
molecular targets for such an approach (Liu, 2005).
To obtain insight into the role of the retinoblastoma susceptibility gene
(Rb; also known as Rb1) in apoptosis,
Caenorhabditis elegans mutants lacking a functional lin-35
RB gene were analyzed. The loss of lin-35 function results in a
decrease in constitutive germ cell apoptosis. Evidence is presented that
lin-35 promotes germ cell apoptosis by repressing the expression of
ced-9, an anti-apoptotic C. elegans gene that is orthologous
to the human proto-oncogene BCL2. Furthermore, the genes
dpl-1 DP, efl-1 E2F and efl-2 E2F were also shown to promote
constitutive germ cell apoptosis. However, in contrast to lin-35,
dpl-1 (and probably also efl-1 and efl-2) promotes germ
cell apoptosis by inducing the expression of the pro-apoptotic genes
ced-4 and ced-3, which encode an APAF1-like adaptor protein
and a pro-caspase, respectively. Based on these results, it is proposed that
C. elegans orthologs of components of the RB tumor suppressor complex
have distinct pro-apoptotic functions in the germ line and that the
transcriptional regulation of components of the central apoptosis machinery is
a critical determinant of constitutive germ cell apoptosis in C.
elegans. Finally, lin-35, dpl-1 and
efl-2, but not efl-1, function either downstream of or in
parallel to cep-1 p53 (also known as TP53) and egl-1
BH3-only were shown to cause DNA damage-induced germ cell apoptosis. These results have implications for the general mechanisms through which RB-like proteins control
gene expression, the role of RB-, DP- and E2F-like proteins in apoptosis, and
the regulation of apoptosis (Schertel, 2007).
The genes egl-1, ced-9, ced-4, and ced-3 play major roles in programmed cell death in Caenorhabditis elegans. To identify genes that have more subtle activities, mutations were sought that confer strong cell-death defects in a genetically sensitized mutant background. Specifically, a screen was performed for mutations that enhance the cell-death defects caused by a partial loss-of-function allele of the ced-3 caspase gene. Mutations were identified in two genes not previously known to affect cell death, dpl-1 and mcd-1 (modifier of cell death). dpl-1 encodes the C. elegans homolog of DP, the human E2F-heterodimerization partner. By testing genes known to interact with dpl-1, roles were identified in cell death for four additional genes: efl-1 E2F, lin-35 Rb, lin-37 Mip40, and lin-52 dLin52. mcd-1 encodes a novel protein that contains one zinc finger and that is synthetically required with lin-35 Rb for animal viability. dpl-1 and mcd-1 act with efl-1 E2F and lin-35 Rb to promote programmed cell death and do so by regulating the killing process rather than by affecting the decision between survival and death. It is proposed that the DPL-1 DP, MCD-1 zinc finger, EFL-1 E2F, LIN-35 Rb, LIN-37 Mip40, and LIN-52 dLin52 proteins act together in transcriptional regulation to promote programmed cell death (Reddien, 2007).
MYC releases early reprogrammed human cells from proliferation pause via retinoblastoma protein inhibition
This study reports that MYC rescues early human cells undergoing reprogramming from a proliferation pause induced by OCT3/4, SOX2, and KLF4 (OSK). ESRG was identified as a marker of early reprogramming cells that is expressed as early as day 3 after OSK induction. On day 4, ESRG positive (+) cells converted to a TRA-1-60 (+) intermediate state. These early ESRG (+) or cell surface marker TRA-1-60 TRA-1-60 (+) cells showed a proliferation pause due to increased p16INK4A and p21 and decreased endogenous MYC caused by OSK. Exogenous MYC did not enhance the appearance of initial reprogramming cells but instead reactivated their proliferation and improved reprogramming efficiency. MYC increased expression of ubiquitin ligase LIN41, which potently suppressed p21 post-transcriptionally. MYC suppressed p16 INK4A. These changes inactivated retinoblastoma protein (RB) and reactivated proliferation. The RB-regulated proliferation pause does not occur in immortalized fibroblasts, leading to high reprogramming efficiency even without exogenous MYC (Rand, 2018).
MYC affects several important biological processes through DNA binding and subsequent transcriptional activation or inhibition of a diverse set of target genes. To define MYC's role in reprogramming, previous reports compared OSK to OSKM (OSK with Myc) reprogramming and found several MYC-dependent correlations. These include enhancement or alteration of OSK binding, promotion of embryonic stem cell (ESC)-like metabolic changes, and enhancement of proliferation. Each of these observed changes may or may not play a major role in the outcome of reprogramming (enhanced colony formation), and we are cognizant of the gap between correlation and causation in this complicated system. To specifically address the significance of a particular function of a multifunctional protein, we need to isolate that particular function and determine its importance. Mutating MYC to isolate a specific biological process (chromatin accessibility versus metabolism versus proliferation) is not feasible: varied biological pathways depend on this transcription factor. In this work, we identified two methods for replacing one particular aspect-enhancement of proliferation-of MYC. This enabled weighing MYC's contribution to proliferation in reprogramming. Both of these methods are discussed in detail here (Rand, 2018).
In addition to the strategies for replacing MYC, identification of ESRG as an early maker of reprogramming proved useful, because it allowed precise definition of cellular and molecular events during the first few days after OSK or OSKM induction. The data demonstrate that OSK elicits two important molecular events in human cells: the initiation of the reprogramming process and the RB-mediated proliferation pause or blockade. MYC does not affect the first event, but it promotes induced pluripotent stem cells (iPSC) generation by escaping the proliferation pause (Rand, 2018).
The first molecular event that OSK elicits is initiation of the reprogramming process, including activating early markers such as ESRG and TRA-1-60. Activation of endogenous retroviruses is crucial for iPSC generation in human cells. ESRG is a lncRNA driven by long terminal repeat (LTR) 7 of a HERV-H group endogenous retrovirus. LTR7 of ESRG contains the binding site of KLF4, suggesting that OSK directly activates its expression. Surprisingly, MYC did not promote this initial process of reprogramming. This is in contrast to a previous report that MYC enhanced the binding of OSK to its targets. Furthermore, even endogenous MYC seemed dispensable for the appearance of ESRG (+) cells. Thus, MYC does not play a crucial role in this early event during human iPSC generation (Rand, 2018).
The other molecular event that OSK elicits is the proliferation pause in early reprograming cells. Few BrdU (+) cells were detected in ESRG (+) cells on day 3 and TRA-1-60 (+) cells on day 4 after induction of OSK. OSK-mediated proliferation pause was attributable to RB activation. It was also found that OSK induced expression of p16 and p53. OSK also induced a transient increase in p21 mRNA levels on day 3. Suppression of these genes by shRNAs induced proliferation. Thus, cooperation of these multiple tumor suppressor gene products is responsible for RB activation and the proliferation pause in early reprogramming cells. Previously, evidence of an oncogene-induced, senescence-like process was detected via transcriptional changes and described as reprogramming-induced senescence. In this paper, the period from day 3 to day 7 is referred to as a proliferation pause or blockade, but there is no objection to alternatively calling the OSK-initiated, p21/p16-driven cessation of proliferation a reprogramming-induced senescence. The two processes are thought to be likely the same. With regards to this process, this work demonstrates the utility of ESRG to define the precise timing and demonstrates the mechanisms by which reprogramming cells are able to overcome this blockade (Rand, 2018).
MYC provided an escape from the proliferation pause elicited by OSK in early reprogramming cells. An important mechanism is the let-7/LIN41/p21 axis. OSK increased transcripts of let-7 family members, but addition of MYC suppressed this increase. LIN41 mRNA expression was induced by OSK in early reprogramming cells. However, the LIN41 protein level remained low due to the increased expression of let-7 that inhibits translation of LIN41 mRNA. MYC, by suppressing let-7 expression, increases LIN41 protein. It was also found that CDKN1A mRNA encoding p21 is the most enriched mRNA binding target in LIN41 pull-down from human ESCs. Furthermore, LIN41 expression in fibroblasts dramatically represses p21 translation. Thus, MYC decreased p21 protein expression, leading to inactivation of RB and initiation of proliferation (Rand, 2018).
Though less dramatic than the effects on the let-7/LIN41/p21 axis, it was additionally found that MYC decreased mRNA and protein levels of p16. When MYC alone is overexpressed, it induces p16 expression, but when it is co-expressed with RAS, MYC suppresses RAS-induced senescence. It is suspected that a similar mechanism operates during reprogramming. In contrast to p16, MYC increased mRNA expression of p53, which should have an inhibitory influence on reprogramming. It is supposed that the suppression of p21 and p16 is sufficient to overcome negative effects of increased p53 (Rand, 2018).
Because a proliferation pause was commonly observed in early reprogramming cells derived from five types of somatic cells, it might be a required step for reprogramming. However, teratoma-derived fibroblasts (TdFs) reprogram without going through a proliferation pause. Furthermore, addition of exogenous LIN41 with OSK allows even somatic cells to avoid the onset of the proliferation pause. Therefore, a proliferation pause is not required in reprogramming; instead, it is a barrier to reprogramming that is common to normal somatic cells (five of five lines) when induced with OSK (Rand, 2018).
TdFs gave a unique opportunity to delineate the roles of MYC in promoting reprogramming. TdFs were highly efficient in iPSC generation, and MYC did not enhance reprogramming. This suggests that the main role or roles of MYC are already achieved in TdFs. An RB-mediated proliferation pause does not occur in TdFs even without exogenous MYC. OSK and OSKM reprogramming efficiencies were equal. In contrast, no metabolic changes were observed in favor of reprogramming in TdFs. The data also suggested that the binding of OSK to their targets was comparable between TdFs and HDFs. Significant downregulation of fibroblast-specific genes was observed in TdFs compared to HDFs, suggesting that this intermediate level of expression may contribute to high reprogramming efficiency of TdFs. However, given that TdFs have an intermediate level of HDF markers, further downregulation of many fibroblast-related genes and upregulation of many ESC-related genes are necessary to form iPSCs. If the previously proposed chromatin accessibility role of MYC significantly affected reprogramming efficiency, it would be expected that TdFs would show a partial improvement upon addition of MYC. In addition, it would be expected that the emergence of early reprogramming cells should be increased regardless of whether the downregulation of the fibroblast genetic program offered an easier route to reprogramming. Neither of these predictions (depending on a significant role for chromatin remodeling by MYC in reprogramming) is true in TdFs. Because proliferation pause-resistant TdFs do not benefit from MYC, it is concluded that the main contribution of MYC in proliferation pause-responsive somatic cells is to overcome proliferation pause rather than to improve reprogramming through other means, such as chromatin accessibility (Rand, 2018).
It was previously shown that LIN41, a cytoplasmic protein and a let-7 miRNA translationally repressed target, dramatically enhanced iPSC generation. EGR1, a transcription factor involved in differentiation, was identified as a target of LIN41. EGR1 protein was upregulated by LIN41 knockdown and LIN41 overexpression suppressed iPSC generation. Based on these data, it was hypothesized that positive action of LIN41 in reprogramming is attributable to suppression of EGR1. However, the current results clearly show that p21 is the most crucial target of LIN41 in promoting iPSC generation. LIN41 enhances the reprogramming of OSK to the same extent as MYC. This is not coincidental, because LIN41 and MYC provide similar outcomes with regard to proliferation pause. OSKL reprogrammed cells subverted the proliferation pause by early and tight translational suppression of p21. OSKM entered the proliferation pause but eventually escaped after downregulation of let-7 miRNA, followed by derepression of endogenous LIN41 protein and subsequently p21 translational silencing. Again, LIN41 likely replaced only one specific biological function out of the various proposed mechanisms of MYC, but it achieved all of MYC's reprogramming enhancement. It is therefore concluded that the significant contribution of MYC is that shared with LIN41-the ability to overcome proliferation pause (Rand, 2018).
It is believed that the metabolic effects described as contingent upon MYC could be viewed as secondary to escape from the proliferation pause. In other words, when OSK is compared to OSKM, one is comparing blocked cells to cells that have escaped proliferation pause, continue to reprogram, and thus are continuing to gain ESC-like attributes (e.g., ESC-like metabolism). Regardless of whether a metabolic contribution is provided by MYC through transcriptional activation of metabolic genes or as a consequence of escape from the proliferation blockade, this study found that an equivalent outcome is obtained by two methods that likely replace only MYC's effect on proliferation. Therefore, a model emphasizing MYC's contribution as facilitating escape from a proliferation pause is favored (Rand, 2018).
In conclusion, the data showed that the main function of MYC during human iPSC generation is to override an RB-mediated proliferation pause in early reprogramming cells. MYC achieves this by suppressing let-7 miRNA and derepressing endogenous LIN41 transcript. This leads to LIN41 protein-dependent translational repression of p21. Exogenous LIN41 fulfills the role of endogenous LIN41 and therefore MYC. In TdFs, the proliferation pause does not operate during reprogramming, resulting in MYC-independent high efficiency of iPSC generation. However, the emergence of early reprogramming cells is inefficient even with MYC or in TdFs. Therefore, the RB pathway is responsible for the proliferation pause, but not the low efficiency of the appearance of early reprogramming cells. Understanding the cause of the low conversion efficiency to the state demarcated by ESRG positivity-an early prerequisite for, but not a guarantee of, inevitable reprogramming-remains an important future task.
The Rb protein suppresses development of an abnormal state of endoreduplication arising after S phase DNA damage. In diploid, S phase cells, the activity of protein phosphatase 2A (PP2A) licenses the stable association of underphosphorylated Rb with chromatin. After damage, chromatin-associated pRb is attracted to certain chromosomal replication initiation sites in the order in which they normally fire. Like S phase DNA damage in Rb-/- cells, specific interruption of PP2A function in irradiated, S phase wt cells also elicits a state of endoreduplication. Thus, PP2A normally licenses the recruitment of Rb to chromatin sites in S phase from which, after DNA damage, it relocalizes to selected replication control sites and suppresses abnormal, postdamage rereplicative activity (Avni, 2003).
Of the four replication initiation sites that were tested (B2-lamin, HSP70, ß-globin, and rRNA), all serve as postdamage Rb targets. Among them are entities that fire early, in mid S phase, and later on in S, suggesting that they are a small but representative set of the replication initiation sites found in the human genome. Given this, one might speculate that, after S phase damage, Rb interacts with a significant fraction of human replication initiation units (Avni, 2003).
The data presented here also suggest that the Rb/replication site interaction is likely related to the replication initiation feature of these elements rather than any function they may have in transcription control. In the absence of this possibility, the evidence points to a rereplication suppression function of Rb mediated through its communication with elements of the replication initiation machinery (Avni, 2003).
What forces govern the interaction of Rb with a given replication initiation site? There is no prior evidence showing that Rb interacts directly with specific DNA structures, although it can certainly do so as part of a complex that contains an active DNA binding element, e.g., an E2F/DP heterodimer. Notably, E2F2 and E2F3 can interact with both oriP and with a replication initiation site near the c-myc promoter. Whether they do so in complex with Rb is unknown. In this regard, Drosophila Rb-E2F complex(es) interact with ORC1 and 2, and Drosophila E2F1 has been identified at a chorion gene region replication origin in Drosophila embryos (Avni, 2003).
As another potential insight into how Rb performs its replication origin control function, Rb can interact with MCM7 and replication factor C, both of which are DNA replication control proteins, and puralpha, a single-strand DNA binding element. Whether any of these interactions underlie Rb/origin refiring suppression in mammalian cells is unclear. However, the participation of E2F in this process in Drosophila led to speculation that Rb complex formation with certain E2F species constitutes part of the relevant mechanism (Avni, 2003).
Passage of normal cells in culture leads to senescence, an irreversible cell cycle exit characterized by biochemical changes and a distinctive morphology. Cellular stresses, including oncogene activation, can also lead to senescence. Consistent with an antioncogenic role for this process, the tumor suppressor pRb plays a critical role in senescence. Reexpression of pRb in human tumor cells results in senescence-like changes, including cell cycle exit and shape changes. Senescence is accompanied by increased expression and altered localization of ezrin, an actin binding protein involved in membrane-cytoskeletal signaling. pRb expression results in the stimulation of CDK5-mediated phosphorylation of ezrin with subsequent membrane association and induction of cell shape changes, linking pRb activity to cytoskeletal regulation in senescent cells (Yang, 2003).
Normal human somatic cells do not divide indefinitely, but rather, have a limited capacity to replicate in culture. The finite replicative lifespan of cells leads to an arrest of cell division by a process termed senescence, clearly distinct from differentiation, in which cells remain metabolically active indefinitely. The irreversible arrest of cell division that accompanies cellular senescence may be tumor suppressive, and escape of cells from senescence accompanies immortalization and oncogenesis. Indeed, a premature senescence is observed following oncogene introduction into primary human cells, and this antiproliferative response must be overcome if cells are to become transformed. Further, senescence may play a significant role in response to cancer therapy (Yang, 2003 and references therein).
Despite this potentially critical role for senescence in tumor formation, knowledge of the biochemical pathways responsible for the acquisition of cellular senescence is rudimentary. The retinoblastoma tumor suppressor protein, pRb, plays a fundamental role in cellular senescence, consistent with a critical role for pRb in the cellular machinery that controls passage from G1 into S phase of the cell cycle. Senescent cells accumulate active pRb, fail to inactivate pRb upon mitogenic stimulation, and consequently cannot enter S phase. Indeed, reintroduction of pRb into Rb-/- tumor cell lines induces senescence, even in cells that do not contain wild-type p53. Similarly, overexpression of p16INK4a can induce senescence in pRb-positive tumor cells. Loss of p16INK4a or pRb function appears to be required for immortalization of at least some human cell types, apparently as an obligate step in preventing senescence (Yang, 2003 and references therein).
RB-transfected SAOS-2 osteosarcoma cells serve as a model system of senescence. Reintroduction of pRb into SAOS-2 cells results in an immediate G1 arrest and subsequent expression of characteristic markers of senescence. The first indication of pRb-induced senescence to be recognized in this system was 'flat cell' formation, typified by an increased cell area and a flattened appearance. This phenotype appears identical to that observed during classical senescence, where a morphological alteration from spindle shape to an enlarged, flattened, and irregular shape is taken as an indicator of the senescent state (Yang, 2003 and references therein).
Despite the universality of morphological changes observed in a wide variety of senescent cells, little is known about the induction of this phenotype nor about its potential contribution to the establishment or maintenance of the irreversible growth arrest that accompanies senescence. Nevertheless, considerable work has clearly indicated a significant role for cell shape in cellular proliferation, largely as a consequence of communication between the cytoskeleton and its associated proteins. One such set of cytoskeletal-associated proteins that has recently emerged as important in proliferation control is the ezrin-radixin-moesin (ERM) family of cytoskeleton-membrane crosslinking proteins, including the related protein NF-2/merlin, an established tumor suppressor. These proteins play a role in the formation of microvilli, cell-cell junctions, and membrane ruffles, and also regulate substrate adhesion and motility. It has most recently become clear that the ERM proteins regulate and respond to proliferative signals, both in a positive and negative manner (Yang, 2003 and references therein).
ERM proteins possess two conserved domains that have been termed N- and C- ERM association domains, or ERMADs. The NH2-terminal domain associates with several transmembrane adhesion molecules, whereas the COOH-terminal domain contains an F-actin binding site. These binding sites are masked in cytoplasmic, inactive ERMs due to an intramolecular N/C-ERMAD interaction. Regulation of ERMs is thought to occur through conformational changes consequent to posttranslational modifications that inhibit association of the N-ERMAD with the C-ERMAD. This scheme has been supported by solution of the crystal structure of the relevant domains of moesin. This work reveals a globular conformation for the N-ERMAD domain and an extended conformation for the C-ERMAD, which mutually mask binding sites for other cellular proteins (Yang, 2003 and references therein).
Phosphorylation has been proposed to regulate ERM activation, since phosphorylation of ERM proteins correlates with their cytoskeletal association. Several observations have suggested that phosphorylation of serine/threonine residues is important for the activity of ERM proteins. Phosphorylation of T567 in ezrin has been found to be critical for conversion of ezrin to the active, open form competent for membrane localization and actin binding. Indeed, structural studies suggest that phosphorylation of T567 would sterically interfere with N-ERMAD/C-ERMAD interactions. Furthermore, induction of apoptosis induces a serine/threonine dephosphorylation of ezrin. This dephosphorylation is essential for the translocation of ezrin from the plasma membrane to the cytoplasm. Thus, regulation of ERM proteins through phosphorylation is likely critical to membrane-cytosekeleton signaling, and this in turn will have a pleiotropic impact on cell shape, motility, and proliferation (Yang, 2003 and references therein).
Ezrin regulation has been linked to pRb function in the senescent phenotype. Ezrin expression increases upon pRb-induced senescence, and more significantly, ezrin becomes membrane associated concomitant with acquisition of the senescent phenotype. This membrane association appears to be the consequence of direct phosphorylation of T235 of ezrin by CDK5, which is activated in response to pRb expression. Phosphorylation of T235 prevents the intermolecular N/C ERMAD association in a manner analogous to and cooperative with phosphorylation of T567, likely allowing ezrin to participate in cytoskeleton-related signaling events germane to senescence (Yang, 2003).
Cellular senescence is an extremely stable form of cell cycle arrest that limits the proliferation of damaged cells and may act as a natural barrier to cancer progression. A distinct heterochromatic structure is descibed that accumulates in senescent human fibroblasts, that is designated senescence-associated heterochromatic foci (SAHF). SAHF formation coincides with the recruitment of heterochromatin proteins and the retinoblastoma (Rb) tumor suppressor to E2F-responsive promoters and is associated with the stable repression of E2F target genes. Notably, both SAHF formation and the silencing of E2F target genes depend on the integrity of the Rb pathway and do not occur in reversibly arrested cells. These results provide a molecular explanation for the stability of the senescent state, as well as new insights into the action of Rb as a tumor suppressor (Narita, 2003).
SAHFs are observed in interphase nuclei and contain the heterochromatin-associated proteins H3 methylated on lysine 9 (K9M-H3) and HP1, exclude histones found in euchromatin (e.g., K9Ac-H3 and K4M-H3), and are not sites of active transcription. SAHFs are distinct from pericentric heterochromatin, and their appearance is accompanied by an increase in HP1 incorporation into senescent chromatin and an enhanced resistance of senescent DNA to nuclease digestion (Narita, 2003).
SAHF formation requires an intact Rb pathway, since expression of E1A, or inactivation of either p16INK4a or Rb, can prevent their appearance. During the initial phases of senescence, Rb might control the nucleation of heterochromatin at specific sites throughout the genome, which then spreads by the action of histone methyltransferases and recruitment of HP1 proteins. HP1 proteins have the capacity to dimerize and may interact to form higher order chromatin structures once a critical mass has been reached. A similar pattern of nucleation and spreading occurs during silencing of the mating type locus in S. pombe, position effect variegation in Drosophila, and X inactivation in mammalian cells, although HP1 proteins do not accumulate on the inactive X. Importantly, SAHF formation correlates precisely with cell cycle exit and the silencing of E2F target genes (Narita, 2003).
Much of what is known concerning the regulation of E2F activity comes from studies examining cell cycle transitions into and out of a quiescent state. These transitions are controlled in a reversible manner, in part, by the competing action of HATs and HDACs on the histones of E2F target promoters. This study compares the physical state and regulation of E2F target genes in quiescent and senescent cells. In both cell states, the amount of K9-aceylated histone H3 that associates with E2F target promoters declines, consistent with the downregulation of transcription that accompanies cell cycle exit. However, in senescent IMR90 cells, histone H3 acetylation is ultimately replaced by methylation at lysine 9, an apparently irreversible modification that prevents acetylation by HATs and is barely observed on E2F-responsive promoters in quiescent cells. Methylated lysine 9 forms a docking site for HP1 proteins and, accordingly, HP1gamma preferentially associates with E2F target promoters in senescent cells. These modifications are predicted to form a 'lock' on the transcription of E2F responsive promoters, making them less accessible to the transcription machinery. Accordingly, several E2F-responsive genes in senescent cells are stably repressed and insensitive to enforced E2F expression relative to quiescent cells. Although it remains to be determined whether every E2F target gene behaves as those studied here, their transition to a heterochromatin-like organization may contribute to the insensitivity of senescent cells to mitogenic signals and the apparent irreversibility of the senescence process (Narita, 2003).
Cellular senescence acts as a potent barrier to tumorigenesis and contributes to the anti-tumor activity of certain chemotherapeutic agents. Senescent cells undergo a stable cell cycle arrest controlled by RB and p53 and, in addition, display a senescence-associated secretory phenotype (SASP) involving the production of factors that reinforce the senescence arrest, alter the microenvironment, and trigger immune surveillance of the senescent cells. Through a proteomics analysis of senescent chromatin, the nuclear factor-kappaB (NF-kapaB) subunit p65 was identified as a major transcription factor that accumulates on chromatin of senescent cells. NF-kappaB acts as a master regulator of the SASP, influencing the expression of more genes than RB and p53 combined. In cultured fibroblasts, NF-kappaB suppression causes escape from immune recognition by natural killer (NK) cells and cooperates with p53 inactivation to bypass senescence. In a mouse lymphoma model, NF-kappaB inhibition bypasses treatment-induced senescence, producing drug resistance, early relapse, and reduced survival. These results demonstrate that NF-kappaB controls both cell-autonomous and non-cell-autonomous aspects of the senescence program and identify a tumor-suppressive function of NF-kappaB that contributes to the outcome of cancer therapy (Chien, 2011).
The tumor suppressor PML (promyelocytic leukemia protein) regulates cellular senescence and terminal differentiation, two processes that implicate a permanent exit from the cell cycle. This study shows that the mechanism by which PML induces a permanent cell cycle exit and activates p53 and senescence involves a recruitment of E2F transcription factors bound to their promoters and the retinoblastoma (Rb) proteins to PML nuclear bodies enriched in heterochromatin proteins and protein phosphatase 1α. Blocking the functions of the Rb protein family or adding back E2Fs to PML-expressing cells can rescue their defects in E2F-dependent gene expression and cell proliferation, inhibiting the senescent phenotype. In benign prostatic hyperplasia, a neoplastic disease that displays features of senescence, PML was found to be up-regulated and forming nuclear bodies. In contrast, PML bodies were rarely visualized in prostate cancers. The newly defined PML/Rb/E2F pathway may help to distinguish benign tumors from cancers, and suggest E2F target genes as potential targets to induce senescence in human tumors (Vernier, 2011).
The retinoblastoma gene family consists of three genes: RB, p107, and
p130. While loss of pRB causes retinoblastoma in humans and pituitary
gland tumors in mice, tumorigenesis in other tissues may be suppressed by p107
and p130. To test this hypothesis, chimeric mice were generated from
embryonic stem cells carrying compound loss-of-function mutations in the
Rb gene family. It was found that Rb/p107- and
Rb/p130-deficient mice are highly cancer prone. It is concluded that, in a
variety of tissues, tumor development by loss of pRB is suppressed by its
homologs p107 and p130. The redundancy of the retinoblastoma proteins in vivo is
reflected by the behavior of Rb-family-defective mouse embryonic
fibroblasts in vitro (Dannenberg, 2004 ).
Condensation and segregation of mitotic chromosomes is a critical process for cellular propagation, and, in mammals, mitotic errors can contribute to the pathogenesis of cancer. This report demonstrates that the retinoblastoma protein (pRB), a well-known regulator of progression through the G1 phase of the cell cycle, plays a critical role in mitotic chromosome condensation that is independent of G1-to-S-phase regulation. Using gene targeted mutant mice, this aspect of pRB function was studied in isolation, and it was shown to be an essential part of pRB-mediated tumor suppression. Cancer-prone Trp53(-/-) mice (the murine version of the p53 gene) succumb to more aggressive forms of cancer when pRB's ability to condense chromosomes is compromised. Furthermore, it was demonstrated that defective mitotic chromosome structure caused by mutant pRB accelerates loss of heterozygosity, leading to earlier tumor formation in Trp53(+/-) mice. These data reveal a new mechanism of tumor suppression, facilitated by pRB, in which genome stability is maintained by proper condensation of mitotic chromosomes (Coschi, 2009).
This study relied on a viable, gene targeted mouse strain in which pRB is mutated to block LXCXE-dependent interactions, such as those with viral oncoproteins and chromatin remodeling enzymes like histone deacetylases. Cells from these mice have limited proliferative control defects, except for the responsiveness to transforming growth factor β (TGF-β) and senescence-inducing stimuli. It was demonstrated that pRB interacts with the Condensin II complex to establish proper chromosome structure. These experiments reveal that condensation defects caused by a deficiency in pRB-LXCXE interactions occur before metaphase, and are unrelated to the ability to regulate G1-to-S-phase progression. Rb1deltaL/deltaL; Trp53-/- mice were used as well as Trp53-/- controls -- both of which are uniformly defective in their response to G1 arrest stimuli such as DNA damage - and oncogene-induced senescence -- to study pRB's role in chromosome condensation in isolation. Rb1deltaL/deltaL; Trp53-/- mice succumb to much more aggressive forms of cancer than p53-deficient controls, and their tumors are characterized by elevated levels of chromosome instability. Furthermore, defective chromosome condensation caused by mutant pRB can accelerate loss of heterozygosity and cancer onset in Trp53+/- mice. This study reveals that participation in mitotic chromosome condensation is an integral aspect of pRB's function as a tumor suppressor protein (Coschi, 2009).
Chromosome instability (CIN) is a common feature of tumor cells. By monitoring chromosome segregation, this study shows that depletion of the retinoblastoma protein (pRB) causes rates of missegregation comparable with those seen in CIN tumor cells. The retinoblastoma tumor suppressor is frequently inactivated in human cancers and is best known for its regulation of the G1/S-phase transition. Recent studies have shown that pRB inactivation also slows mitotic progression and promotes aneuploidy, but reasons for these phenotypes are not well understood. This study describes the underlying mitotic defects of pRB-deficient cells that cause chromosome missegregation. Analysis of mitotic cells reveals that pRB depletion compromises centromeric localization of CAP-D3/condensin II and chromosome cohesion, leading to an increase in intercentromeric distance and deformation of centromeric structure. These defects promote merotelic attachment, resulting in failure of chromosome congression and an increased propensity for lagging chromosomes following mitotic delay. While complete loss of centromere function or chromosome cohesion would have catastrophic consequences, these more moderate defects allow pRB-deficient cells to proliferate but undermine the fidelity of mitosis, leading to whole-chromosome gains and losses. These observations explain an important consequence of RB1 inactivation, and suggest that subtle defects in centromere function are a frequent source of merotely and CIN in cancer (Manning, 2010).
Loss of G1/S control is a hallmark of cancer, and is often caused by inactivation of the retinoblastoma pathway. However, mouse embryonic fibroblasts lacking the retinoblastoma genes RB1, p107, and p130 (TKO MEFs) are still subject to cell cycle control: Upon mitogen deprivation, they enter and complete S phase, but then firmly arrest in G2. This study shows that G2-arrested TKO MEFs have accumulated DNA damage. Upon mitogen readdition, cells resume proliferation, although only part of the damage is repaired. As a result, mitotic cells show chromatid breaks and chromatid cohesion defects. These aberrations lead to aneuploidy in the descendent cell population. Thus, the results demonstrate that unfavorable growth conditions can cause genomic instability in cells lacking G1/S control. This mechanism may allow premalignant tumor cells to acquire additional genetic alterations that promote tumorigenesis (van Harn, 2010).
The miR-17~92 cluster, consisting of seven miRNAs transcribed as a polycistronic message from an intron of the C13-25orf locus, is a potent microRNA-encoding oncogene. This study show sthat miR-17~92 synergizes with loss of Rb family members to promote retinoblastoma. miR-17~92 genomic amplifications was observed in murine retinoblastoma and high expression of miR-17~92 was seen in human retinoblastoma. While miR-17~92 is dispensable for mouse retinal development, miR-17~92 overexpression, together with deletion of Rb and p107, led to rapid emergence of retinoblastoma with frequent metastasis to the brain. miR-17~92 oncogenic function in retinoblastoma was not mediated by a miR-19/PTEN axis toward apoptosis suppression, as found in lymphoma/leukemia models. Instead, miR-17~92 increased the proliferative capacity of Rb/p107-deficient retinal cells. Deletion of Rb family members led to compensatory up-regulation of the cyclin-dependent kinase inhibitor p21Cip1. miR-17~92 overexpression counteracted p21Cip1 up-regulation, promoted proliferation, and drove retinoblastoma formation. These results demonstrate that the oncogenic determinants of miR-17~92 are context-specific and provide new insights into miR-17~92 function as an RB-collaborating gene in cancer (Cronkite, 2011).
Inactivation of RB is one of the hallmarks of cancer, however gaps remain in understanding how RB-loss changes human cells. This study showed that pRB-depletion results in cellular reprogramming: how RB-depletion altered the transcriptional, proteomic and metabolic output of non-tumorigenic RPE1 human cells was quantitatively measured. These profiles identified widespread changes in metabolic and cell stress response factors previously linked to E2F function. In addition, a number of additional pathways was found that are sensitive to RB-depletion, that are not E2F-regulated and that may represent compensatory mechanisms to support the growth of RB-depleted cells. To determine whether these molecular changes are also present in RB1(-/-) tumors, these results were compared to Retinoblastoma and Small Cell Lung Cancer data, and widespread conservation of alterations found in RPE1 cells was identified. To define which of these changes contribute to the growth of cells with de-regulated E2F activity, how inhibiting or depleting these proteins affected the growth of RB1(-/-) cells and of Drosophila E2f1-RNAi models in vivo was assayed. From this analysis, key metabolic pathways were identified that are essential for the growth of pRB-deleted human cells (Rajasekaran, 2021).
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