Download DOES THE RIBOSOME TRANSLATE CANCER?

REVIEWS
DOES THE RIBOSOME TRANSLATE
CANCER?
Davide Ruggero and Pier Paolo Pandolfi
Ribosome biogenesis and translation control are essential cellular processes that are governed at
numerous levels. Several tumour suppressors and proto-oncogenes have been found either to
affect the formation of the mature ribosome or to regulate the activity of proteins known as
translation factors. Disruption in one or more of the steps that control protein biosynthesis has
been associated with alterations in the cell cycle and regulation of cell growth. Therefore, certain
tumour suppressors and proto-oncogenes might regulate malignant progression by altering the
protein synthesis machinery. Although many studies have correlated deregulation of protein
biosynthesis with cancer, it remains to be established whether this translates directly into an
increase in cancer susceptibility, and under what circumstances.
NUCLEOLUS
The nucleolus is a suborganelle
of the nucleus where rRNA is
transcribed, processed and
assembled into ribosomal
subumits. Ribosomal proteins
are added to rRNAs within the
nucleolus.
Molecular Biology Program,
Department of Pathology,
Memorial Sloan-Kettering
Cancer Center,
Sloan-Kettering Institute,
1275 York Avenue, New York,
New York 10021, USA.
Correspondence to P. P. P.
e-mail:
[email protected]
doi:10.1038/nrc1015
NATURE REVIEWS | C ANCER
The production of mature ribosomes that are competent to translate cellular mRNAs requires a multistep
process that is highly coordinated in eukaryotic cells.
The ribosome, which is the central protein synthesis
factory of the cell, is often viewed as a finely tuned
machine that functions as a static, reliable component
of higher-order cellular processes. In fact, the ribosome has the overwhelming burden of correctly and
efficiently producing all proteins in the cell. Although
it has long been known that, in cancer cells, components of the translation machinery are deregulated or
misexpressed, their role in tumorigenesis has largely
been overlooked. For example, as early as 1970,
changes in the NUCLEOLUS were recognized as a reliable
marker of cellular transformation1. However, these
findings were dismissed as non-consequential to the
overall transformation process. After all, how could the
ribosome, which is required for such a crucial cellular
function, be tweaked or perturbed without causing
death to the cell?
An exciting body of research is now challenging
this concept. In the past, the protein synthesis
machinery was viewed as a static entity, but it is now
becoming clear that its activity is highly regulated. In
fact, several proto-oncogenes and tumour suppressors have recently been shown to directly regulate
ribosome production or the initiation of protein
translation, or both (TABLE 1). These findings raise the
possibility that alterations in components of the protein synthesis machinery and aberrant regulation of
translation can promote cellular transformation. The
goal now is to determine how and to what extent ribosome function is directly modulated by tumour suppressors and oncogenes, and whether this represents a
cause or consequence of cancer progression.
Mutations in genes that encode proteins that are
directly involved in ribosome biogenesis have also been
associated with cancer and human disease. The DKC1
gene is mutated in patients with dyskeratosis congenita,
a disease that is characterized by premature ageing and
an increased susceptibility to cancer2,3. DKC1 encodes
dyskerin, a pseudouridine synthase that mediates posttranscriptional modification of ribosomal RNA.
Mutations in the gene that encodes ribosomal protein
S19 have been identified in another syndrome that is
characterized by increased susceptibility to cancer —
Diamond–Blackfan anaemia4. But do mutations in
these genes directly result in impaired mRNA translation? Is this a quantitative or qualitative translation
problem? How can defects in protein synthesis result
in a specific disease phenotype or carcinogenesis?
Although there are many questions to be answered, it is
now becoming evident that regulation of ribosome
function can be lost in cancer cells.
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All roads lead to rRNA synthesis
Cell growth and proliferation are associated with changes
in the rate of ribosome production. During G1, an
increase in rRNA synthesis and ribosome assembly is a
prerequisite for increased protein synthesis during
S phase5. Furthermore, downregulation in ribosome
activity or ribosome formation, or both, could be
required during M phase to ensure proper exit from the
cell cycle6,7. Therefore, an important relationship exists
between the cell cycle and ribosome production. This balance is maintained in the cell through key checkpoints,
which ensure that translation of mRNAs occurs at appropriate levels and during a specific window of the cell cycle.
As will be discussed below, in cancer cells this balance can
be broken, resulting in deregulation of rRNA synthesis.
The synthesis of rRNA is the first event in ribosome
biogenesis. It is dependent on RNA polymerase I (Pol I)
regulation of rDNA transcription in the nucleolus (BOX 1).
The synthesis of rRNA in the cell can be induced by
extracellular stimuli at moments when a cell needs to
grow and proliferate. The concept of regulated rRNA
synthesis was originally shown in cells in which deprivation of a single amino acid resulted in a rapid termination of rRNA synthesis8. Since then, many other reports
have shown that the initiation of rRNA transcription is
tightly linked to cell-cycle progression. Synthesis of
Summary
• Ribosome biogenesis and translation are regulated at multiple levels and are associated
with accurate cell growth and proliferation. The loss of key checkpoints during protein
synthesis might contribute to the initiation and progression of cancer.
• During specific phases of the cell cycle, the synthesis of rRNA, as well as components of
the protein synthesis machinery, is initiated by the phosphorylation of key
transcription factors that regulate polymerase I (Pol I) and Pol III activity, respectively.
This tight link between cell-cycle progression and protein synthesis exists to ensure
accurate cell growth and proliferation, which might be lost in cancer cells.
• p53 and retinoblastoma (RB) repress Pol I and Pol III transcription. In cancer cells,
which harbour inactivating mutations in these tumour suppressors, deregulation of
Pol I and Pol III activity might contribute to tumorigenesis.
• Several ribosomal proteins are overexpressed in a variety of tumours. It remains to be
determined whether this represents a cause or consequence of tumour formation.
Increased phosphorylation of the S6 ribosomal protein is thought to result in enhanced
translation of specific mRNAs. This raises the possibility that deregulation of
ribosomal proteins in tumours might affect the translation of specific target mRNAs.
• MYC and PTEN act as master regulators of ribosome biogenesis and translation
control. Their deregulation in tumour cells increases the expression and activity of
components of the translation apparatus. It remains to be determined which of the
downstream targets of MYC and PTEN involved in controlling protein synthesis are
directly responsible for tumour susceptibility.
• Further investigation will be needed to clarify to what extent deregulation in total or
specific translation of mRNAs contributes to tumorigenesis. Although mutations in
genes that are directly responsible for ribosome biogenesis, such as those encoding the
ribosomal protein S19 and DKC1 (the enzyme that modifies rRNA), have been found
in cancer susceptibility syndromes, the molecular mechanisms by which these proteins
cause cancer remain largely unknown.
• Components of the translation machinery that are overexpressed or deregulated in
cancer cells could represent targets for cancer therapy. The macrolide rapamycin,
which affects the translation machinery, has already been used in clinical trials as a
tumour inhibitory agent.
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rRNA is maximal in S and G2 phases, repressed in mitosis and increased in G1 (REFS 7,9,10). These fluctuations in
cell-cycle-mediated rRNA synthesis are dependent on
the activity of Pol I. The transcription factor UBF
(upstream binding factor) is a key regulator of rRNA
synthesis owing to its ability to modulate Pol I transcriptional activity11–13. Furthermore, several proto-oncogenes and tumour suppressors directly regulate rRNA
synthesis by potentiating or repressing UBF activity,
respectively (FIG. 1).
UBF binds to two regions of the rDNA promoter —
the upstream control element (UCE) and the core —
which are also recognized by Pol I (REFS 14–16).
Phosphorylation of UBF upregulates rRNA synthesis in
response to external stimuli (FIG. 1). The amount of prerRNA in cells that express a mutant non-phosphorylatable form of UBF is twofold lower than in cells that
express wild-type UBF, with a concomitant reduction in
cell growth17. Different kinases phosphorylate UBF at
serine and threonine residues positioned at the carboxyterminal and internal regions of the protein. The modification of UBF predominately affects its ability to bind
to DNA and/or to mediate protein–protein interactions
that are required for assembly of an active initiation
complex18–21. Importantly, phosphorylation of UBF is
directly dependent on mitogen stimuli and cell-cycle
progression, and, in quiescent cells, UBF is hypophosphorylated and transcriptionally inactive22–24. UBF
phosphorylation therefore represents a point of convergence for many cell-cycle-regulated proteins in modulated rRNA synthesis. It could also represent a potential
target for proto-oncogenes, some of which have been
shown to directly regulate UBF activity.
The first protein identified to regulate UBF activity
was casein kinase II (CKII)23 — a serine threonine kinase
that is overexpressed in many cancers, including
leukaemias and solid tumours. CKII has been proposed
to contribute to tumorigenesis through direct interactions with the cell-cycle machinery. In addition, it phosphorylates UBF at its carboxyl terminus and thereby
regulates rDNA transcription. Subsequent studies have
identified other UBF kinases that are also deregulated
in cancer and similarly affect rRNA synthesis. The
complexes that are formed by G1-specific cyclin-dependent kinases and cyclins — CDK4–cyclin-D1 and
CDK2–cyclin-E — directly phosphorylate UBF at serine
484 and 388, facilitating the interaction between UBF and
Pol I and so controlling rRNA synthesis in a cell-cycleregulated manner17,20. The CDK–cyclin complex is overexpressed by human neoplasias. Transgenic mice that
express proteins that mimic the human mutant forms
also develop tumours 25,26.
Would alterations in rRNA synthesis have a consequence for cancer progression, and, if so, what would
be the underlying mechanism? The above-mentioned
kinases all mediate phosphorylation of UBF in a specific window of the cell cycle. Although all these
kinases are overexpressed in cancer, the exact implications of upregulation in rRNA synthesis on the transformation process are unclear. If cancer cells have lost
cell-cycle control, upregulation in rRNA synthesis
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Table 1 | Translation control and cancer
Gene
Syndrome or tumour type
Function in protein synthesis
TP53
Wide range of tumours
Regulates Pol I and Pol III activity
RB
Retinoblastoma; a wide
range of tumours
Regulates Pol I and Pol III activity
NPM
Haematological malignancies;
overexpressed in a wide
range of tumours
Ribosome biogenesis
PTEN
Cowden disease; a wide
range of tumours
Regulates mTOR kinase;
important for phosphorylation of
S6 and 4EBP1
Ribosomal protein
Overexpressed in a wide
range of tumours
Ribosome biogenesis
S19
Diamond–Blackfan anaemia;
haematological malignancies;
myelodysplastic syndrome
Ribosome biogenesis
S6
NA
Translation of 5′ TOP mRNAs
DKC1
Dyskeratosis congenita;
haematological malignancies;
carcinomas
Modification of rRNA
mTOR, mammalian target of rapamycin; NA, not applicable; NPM, nucleophosmin; Pol, polymerase;
RB, retinoblastoma; TOP, terminal oligopyrimidine tract.
would simply be a readout of upregulated cellular
proliferation. In neoplasia, however, upregulation of
UBF activity has been shown to arise from direct
extrastimulatory stimuli. This represents an instance
in which upregulation of rRNA synthesis does not
simply reflect cell-cycle progression, but, rather, UBF
serves as a stimulus that might directly activate the
translational machinery to initiate tumorigenesis.
Little is known about the molecular mechanism by
which an extracellular cue could directly regulate Pol I
activity and augment rRNA synthesis.
The first indication of the way in which specific
growth factors could regulate rRNA synthesis came
from the finding that epidermal growth factor (EGF)
signalling via extracellular-signal-regulated kinase
(ERK) activation could directly regulate UBF activity21.
The ERK1/2 kinases were shown to phosphorylate UBF
at two threonine residues, which resulted in an immediate upregulation of endogenous ribosomal transcription. This finding places ribosomal transcription under
the direct control of extracellular growth signals. Given
the finding that ERK is upregulated in many cancers27,
the early cellular response to upregulate rRNA synthesis
might prove to be a mechanism by which ERK mediates
cellular transformation.
The phosphorylation of UBF by ERK might be one
mechanism by which rRNA synthesis is controlled by
extracellular growth signals. However, given that shutdown of rRNA synthesis during mitosis and early G1
was shown to be due to the inactivation of UBF by its
dephosphorylation10, the ability of kinases to upregulate
rRNA synthesis would be limited to a specific window
of the cell cycle. The determination of the phosphatase
that is directly responsible for mediating the mitotic
repression of UBF activity will undoubtedly shed light
on an important checkpoint within the cell, as this
phosphatase would prevent inappropriate ribosome
biogenesis during cell division.
NATURE REVIEWS | C ANCER
Some evidence exists to indicate that the tumoursuppressor protein phosphatase (PP2A) mediates
dephosphorylation of UBF10. Deregulation of this phosphatase in cancer cells could have an even more profound effect in disrupting the coordination between
rRNA synthesis and cell-cycle progression. Fluctuations
in rRNA synthesis, which occur in various conditions
that affect cell growth, also correlate with the activity of
the transcription initiation factor TIF-IA. Mammalian
TIF-IA serves as a bridge between Pol I and the pre-initiation complex on the rDNA promoter28,29 (FIG. 1).
Remarkably, cellular lysates from cells that arrest in the
cell cycle by confluent inhibition, and are starved of
amino acids or treated with cyclohexamide, are incapable of efficiently transcribing rDNA. Addition of
recombinant TIF-IA to cells, however, completely
restores transcriptional activity29. Given that TIF-IA is
phosphorylated at multiple sites by different extracellular signals29, the identification of the kinases that mediate its phosphorylation will be an exciting avenue for
future research. It would shed light on how mitotic
stimuli, which are relevant in cancer progression,
directly regulate rRNA synthesis.
Regulation of protein synthesis by RB and p53
The family of retinoblastoma proteins (RB) have also been
shown to regulate rRNA synthesis through UBF (FIG. 1).
The RB gene is frequently disrupted in human tumours.
Its function as tumour suppressor has been attributed to
its ability to regulate the cell cycle, which involves modulation of the transcriptional activity of E2F 30. In addition,
several findings indicate that RB regulates cell growth and
proliferation by restricting the production of rRNA11. It is
accomplished by the direct association of RB with UBF.
This interaction prevents UBF from recruiting other
cofactors that are required for Pol I activity, such as
TIF-IB/SL1(REFS 31,32).Another RB-family member, p130,
also represses rRNA transcription through its ability to
bind and inactivate UBF 33.
Three lines of evidence have highlighted the potential importance of the interaction of UBF and RBfamily members in controlling cell proliferation.
Immunofluorescence studies have shown that RB
accumulates in the nucleolus when cells differentiate
or downregulate Pol I transcription, or both 11,34.
Overexpression of RB can repress the rDNA promoter
both in vitro and in vivo. Furthermore, rRNA concentrations are abnormally raised in cells that lack the
entire RB family of proteins, or a combination of RB
and p130 (REF. 33). Cells that lack both RB and p130 do
not show significant differences in cell-cycle progression. Therefore, an upregulation of rRNA in this case
cannot be explained simply as a consequence of
increased cell-cycle progression. These results show that
the effects of RB on cell growth can be discriminated
from its ability to regulate UBF activity.
Similar to RB and p130, the tumour suppressor p53
has been shown to repress Pol I transcription by directly
interfering with the assembly of a protein complex that
is required for transcriptional initiation on the rRNA
promoter35 (FIG. 1). Wild-type p53 can suppress Pol I
VOLUME 3 | MARCH 2003 | 1 8 1
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transcription in cotransfection assays, and p53-null cells
display increased Pol I transcriptional activity compared
with wild-type cells35. Because RB-inactivating mutations
also occur in tumour cells that harbour p53 mutations36,
the combined effect of both mutations on Pol I activity
might have a synergistic effect on tumour progression.
Although beyond the scope of this review, p53 and
RB-family members have also been shown to control
RNA polymerase III (Pol III) transcription37–39 (FIG. 1).
Pol III is responsible for synthesizing various small stable RNAs that include components of ribosome, such
as 5′SrRNA, as well as tRNA. In p53 and RB-null cells,
Pol III transcription is increased with respect to wildtype cells. Both of these tumour suppressors negatively
regulate Pol-III-mediated transcription through direct,
inactivating interactions with TF-IIIB — a co-activator
complex that is responsible for Pol-III-mediated transcription. Therefore, loss of RB and p53 in tumours
could result in increased cell proliferation through
aberrant upregulation in essential components of the
protein synthesis machinery.
Several tumour-associated RB mutations specifically
interfere with the ability of RB to regulate Pol I or Pol III
activity, or both. A RB mutation found in small-cell lung
carcinoma that results in a single amino acid change
prevents RB interaction with UBF and repression of Pol I
transcription11,40. In addition, several mutations in RB
lead to deletions of regions that control Pol III activity,
and the ability of RB to repress Pol III activity was
shown to be lost in these tumours41. So, the ability of RB
to directly suppress the level of protein synthesis by controlling the availability of tRNA and rRNA might be
compromised in several tumour lesions.
How can increases in Pol I and Pol III activity be
directly linked to cancer? It has been known for more
than 25 years that the rate of cellular proliferation and
growth is proportional to the rate of protein synthesis42,43.
Translation is dependent on the availability of synthesized
tRNA and rRNA. Both RB-family members and p53
could exert some aspects of their tumour-suppressor
properties through their ability to suppress Pol I and Pol
III transcriptional activity and, subsequently, the level of
protein synthesis. In addition, the upregulation in cancer
of the UBF kinases described above could stimulate
rRNA synthesis and contribute to their oncogenic properties. Disruption of protein synthesis control could render the cells more susceptible to deregulated cell growth
and proliferation. It remains to be determined to what
extent deregulation of Pol I and Pol III transcription
contributes to tumour initiation or progression, or both.
Box 1 | Making ribosomes
UCE Core
ψ and CH3 modification
SnoRNPs
ψ ψ ψ ITS 1 ψ ITS 2 ψ
18S
5.8S
45S
5′ETS
CH3 CH3
ψ
ψ ψ
28S
CH3
CH3 CH3 CH3
3′ETS
41S
36S
32S
RNases
18S
5.8S
ψ ψ ψ
28S
S+
CH3
ψ ψ
18S
CH3
CH3
ψ
ψ
ψ
ψ
ψ
CH3
5.8S
ψ
5S
CH3
ψ
L
S
ψ
+ CH3
28S
CH
3
Ribosomal
proteins
L
12S
ψ
| MARCH 2003 | VOLUME 3
UBF
TIF-IB/SL1
ψ
182
POL I
TIF-IA
3 ψ
CH
All ribosomal RNAs, with the exception of the 5S, are
transcribed as a polycistronic transcript known as preribosomal RNA (pre-rRNA or 45S) in the nucleolus46.
Transcription of rDNA is dependent on three basal
transcription factors: the ‘selectivity complex’ (SL1 or TIFIB), the HMG1 box architectural upstream binding factor
(UBF) and the DNA-dependent RNA polymerase I (Pol I).
Both UBF and SL1 make direct contacts with a 150-bp
region of DNA in the rRNA gene promoter, which contains
two sequence elements known as the upstream control
element (UCE) and the core. In the case of UBF, it probably
binds to these promoter elements as a dimer. UBF is
thought to bind the promoter first, enabling subsequent
recruitment of SL1 and Pol I. The initiation factor TIF-IA is
also part of this complex.
Concomitant with rRNA transcription, the rRNA
sequences are extensively modified134. Specifically, a large
family of small nucleolar RNAs (snoRNAs) guides the sitespecific conversion of uridine to pseudouridine (Ψ) in
rRNA. This is accomplished through direct base-pairing of
snoRNAs with specific rRNA sequences, leaving a single
uridine exposed to the enzymatic activity of dyskerin, the
pseudouridine synthase that mediates the modification of
this residue. In addition, other snoRNAs also guide the
formation of 2′-0-methylated nucleosides (-CH3) in rRNA.
The pre-rRNA precursor is then cleaved at specific sites by
RNases to produce a series of characteristic intermediates
(41S), and finally mature rRNAs — 18S, 5.8S and 28S.
During rRNA processing, the rRNA species must
associate with more than 70 ribosomal proteins, as well as
the 5S rRNA in the nucleolus, to form the small (S, 40S)
and the large (L, 60S) ribosomal subunits, which are
assembled and transported to the cytoplasm to initiate
protein synthesis.
CH3
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5S
tRNA
snRNAs
POL III
RB
p53
TFIIIB TFIIIC2
P
TIF-1A
P
POL I
rRNA
TIF-1B/SL1
UBF
18S
5.8S
28S
P P
ERK
CDK2/4
CKII
Figure 1 | Regulation of Pol I and Pol III activity by tumour
suppressors and proto-oncogenes. The tumour
suppressors retinoblastoma (RB) and p53 negatively regulate
RNA polymerase (Pol I) and Pol III complex activity. Pol I activity
transcribes rRNA, whereas Pol III transcribes a class of small
RNAs that includes the 5S rRNA, small nuclear RNAs (snRNAs)
and tRNA. Both RB and p53 interfere with the assembly of
transcriptional machinery on the rRNA promoter by inhibiting
the interaction between TIF-IB/SL1 and upstream binding
factor (UBF). In the case of p53, the interaction of SL1 and UBF
is destabilized by the ability of p53 to bind to SL1, whereas RB
directly interacts with UBF. RB and p53 also bind and repress
the TFIIIB transcription factor, thereby negatively regulating Pol
III activity. Therefore, mutations that inactivate the tumoursuppressor activity of RB or p53, or both, result in aberrant
upregulation in essential components of the protein synthesis
machinery and increase ribosome biogenesis, leading to
enhanced mRNA translation rates. UBF activity is also tightly
regulated at the level of its phosphorylation (P). The carboxyterminal acidic region of UBF is phosphorylated by cyclindependent kinase 2/4 (CDK2/CDK4) and casein kinase II
(CKII), and this has been correlated with enhanced binding of
UBF to SL1. A rapid activation of endogenous rRNA by
epidermal-growth-factor signalling is mediated by direct
phosphorylation of UBF by the mitogen-activated protein
(MAP)-kinase ERK (extracellular signal-regulated kinase). Given
that the CDK and cyclin complex that phosphorylates UBF, as
well as ERK, are overexpressed in human neoplasias, an
increase in UBF modification could increase rRNA transcription
and promote transformation. Phosphorylation of the initiation
factor TIF-IA after stimulation by various extracellular stimuli
correlates with an increase in growth-dependent regulation of
Pol I activity, and the kinases that initiate this phosphorylation
are likely to regulate rRNA synthesis on stimulation with
mitogenic stimuli.
CRYO-ELECTRON MICROSCOPY
A technique that is used to study
the three-dimensional structures
of biomolecules. A sample is
rapidly frozen in liquid helium
to preserve its structure and then
examined in the frozen, hydrated
state in a cryoelectron
microscope.
NATURE REVIEWS | C ANCER
Although compelling biochemical evidence
indicates that many proto-oncogenes and tumour
suppressors interact directly with components of
the Pol I and Pol III transcription complex, the
functional consequences of increased rRNA synthesis on translation or in the increased production of
functional ribosomes is lacking. Are translation
rates upregulated in all cancer cells? Does specificity exist in the type of mRNAs that show a potential increase in translation following an increase in
Pol I and/or Pol III activity? Furthermore, given
that many of the tumour suppressors and protooncogenes that regulate rRNA synthesis have additional cellular functions, further research is
required to clarify the consequences of increased
Pol I and Pol III activity in cancer cells.
Some indication of the effects of increasing ribosome
synthesis comes from recent characterization of the
Drosophila tumour suppressor mutant brain tumor
(brat). Homozygous brat mutant flies die of an enlarged
brain that is up to eight times bigger than the normal
size, and these tumours consist of malignant optic neuroblasts that have the potential to metastasize44. The brat
gene encodes a protein that regulates rRNA synthesis in
the fly. The Caenorhabditis elegans homologue of brat,
ncl-1, has been shown to directly negatively regulate both
Pol I and Pol III transcription45, and brat is able to rescue
the phenotype of ncl-1 mutants. This indicates that the
function of this protein is conserved during evolution.
The brat-mutant tumour phenotype is associated
with an increase in rRNA synthesis, although little is
known about the precise mechanism by which Brat
regulates Pol I transcription. The brat-mutant cells
are larger than wild-type cells and they have enlarged
nucleoli, which are associated with an increase in
total rRNA production. These results provide evidence that the tumour phenotype of brat mutants
could be caused by excessive cell growth and ribosome synthesis. The association of cell growth and
increased ribosome function is commonly associated
with overproduction of the translation apparatus (see
below). It remains to be determined whether this is
merely a correlation, as cells that produce more ribosomes are larger to enable them to accommodate
their increased protein load, or whether increased cell
size actually promotes tumour development.
Ribosomal proteins and tumorigenesis
More than 70 ribosomal proteins (r-proteins) must
assemble with the rRNAs in the nucleolus to form the
small and the large ribosomal subunits 40S and 60S46
(BOX 1). Several ribosomal proteins possess conserved
domains that are important for their interaction with
rRNA47. In the past, it was assumed that r-proteins stabilize specific rRNA structures in mature ribosomal subunits and promote correct folding of rRNAs during
ribosome assembly. The recent solution of the ribosome
structure by X-ray crystallography and CRYO-ELECTRON
MICROSCOPY has improved our understanding of the specific roles of different regions of rRNA, as well as the
function of r-proteins during the steps that lead to
protein synthesis48,49. These data revealed that many
r-proteins might function as RNA chaperones not only
during the assembly of the ribosome particles, but also
in the stabilization of important domains of the rRNA,
such as the peptidyl transferase center of the large subunit. In addition, r-proteins could coordinate the interaction between the ribosome and mRNA, as well as with
initiation and elongation factors. Why should the activity of r-proteins interest the cancer biologist? Alterations
in the expression of r-proteins seem to be consistently
associated with tumorigenesis. In fact, many r-proteins
that belong to the small (S) and the large (L) ribosomal
subunit are overexpressed in cancer cell lines, as well as
in primary tumours50–52. R-proteins are markers for general cellular transformation, as single and multiple
groups of proteins are overexpressed by solid tumours
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Growth factors,
mitogen stimuli
PI3K
PTEN
?
PDK1
X
AKT
5′ TOP mRNA
translation
P
S6
Elongation factors
Ribosomal proteins
Ribosome biogenesis factors
mTOR
S6K
Rapamycin
?
TSC1
TSC2
Figure 2 | Regulation of protein synthesis by the PI3K signal-transduction pathway.
The activation of the phosphotidylinositol 3-kinase (PI3K) signal-transduction pathway by
growth factors and mitogenic stimuli results in increased translation of a subset of mRNAs
that contain a terminal oligopyrimidine tract (TOP) in their 5′ untranslated region (UTR). The
5′ TOP genes encode many components of the translation apparatus, including ribosomal
proteins and elongation factors. Three different signal-transduction pathways downstream of
PI3K activation have been proposed to account for regulation of 5′ TOP gene expression
following mitogenic stimuli. In this first pathway, direct activation of the kinase mTOR
(mammalian target of rapamycin) by AKT results in increased S6 kinase (S6K) activity and,
subsequently, the phosphorylation of the ribosomal protein S6. Activation of S6 by
phosphorylation leads to an upregulation in the translation of selective messengers, 5′ TOP
genes, through an unknown mechanism. It has also been shown that a second signaltransduction pathway exists whereby PDK1 can directly phosphorylate S6K, leading to
activation of S6. Deregulation of S6 phosphorylation could therefore contribute to
tumorigenesis elicited by activation of the PI3K signal-transduction pathway in cancer. The
tumour suppressor tuberous sclerosis complex-2 (TSC2), which exists as a heterodimeric
complex with TSC1, acts as an antagonist of S6K activation. It has been proposed that
TSC1/2 can either inhibit mTOR activity by preventing its phosphorylation or inhibit S6K
phosphorylation through a poorly defined pathway. Furthermore, AKT can also inhibit the
formation of the TSC1/2 complex by phosphorylating TSC2 and thereby disrupting the activity
of this S6K antagonist on stimulation with mitogenic stimuli107,135,136. Cancer cells that lose
TSC1/2 function therefore might also show increased 5′ TOP mRNA production, leading to
increased uncontrolled protein synthesis. It has recently been shown that a third signaltransduction pathway could also exist, whereby the regulation of 5′ TOP mRNA translation
would be dependent on the PI3K signal-transduction pathway, but independent of both S6K
and phosphorylation of S6. This pathway involves unidentified protein components (X). Given
that most 5′ TOP genes encode proteins that can upregulate protein synthesis, a feedback
loop might exist by which an increase in S6 phosphorylation results in an upregulation in total
protein synthesis in the cell. Rapamycin, a specific inhibitor of the mTOR kinase, is currently in
clinical trials for its growth/tumour-suppressive activity.
and by leukaemia cells53–57. How could alterations in
r-protein expression lead to a defect in ribosome function, and, more importantly, to alterations in cellular
proliferation?
The synthesis of r-protein genes is a coordinated
process, leading to the precise equimolar production
of 78 proteins58. Investigations into the consequences
of deletions in specific r-proteins have been conducted mainly on lower eukaryotic organisms such as
yeast. Gene-targeting experiments in yeast have
shown that the depletion of a single r-protein such as
L16, results in a decrease of the 60S ribosomal
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subunit, and that this is associated with a reduction
in polyribosomes and a defect in cellular growth 59.
Disrupting expression of only one r-protein can
therefore shut down ribosome production.
The control of ribosome biogenesis by single r-proteins has been validated for other r-proteins in addition
to L16, and it has also been shown in other model
organisms. Mutations in individual r-proteins in
Drosophila lead to a class of mutants collectively
known as minute. Minute flies are characterized by
reductions in body size, diminished fertility and recessive lethality 60. Direct and indirect evidence support
the fact that minute cells contain fewer ribosomes and
so have a reduced capacity for protein synthesis61.
Reductions in protein synthesis could reduce cell
growth and proliferation.
Verification of this hypothesis in higher organisms
has been partially validated by experiments that involve
the conditional deletion of the 40S r-protein, S6, in
mice. This results in defective ribosome biogenesis and
reduced cell proliferation62. The ribosomal protein S6 is
of particular interest, due to the fact that its phosphorylation is tightly regulated by extracellular signalling
pathways that are deregulated in cancer cells (see
below). S6 phosphorylation has been associated with
translation of a specific class of mRNAs termed TOP (a
terminal oligopyrimidine tract in the 5′ untranslated
region (UTR)) mRNA63–65. This class of mRNAs
includes ribosomal proteins, elongation factors 1A1
and 2 (EEF1A1 and EEF2) and several other proteins
that are involved either in ribosome biogenesis or in
translation control (FIG. 2).
TOP mRNAs are regulated at the translation level as
they are shifted from polysomes (active translational
ribosomes) in growing cells into mRNP (inactive translational particles) in quiescent cells66. The proteins
encoded by TOP genes might themselves act as protooncogenes. For example, EEF2 — an isoform of EEFIA
— is amplified in primary ovarian tumours, and its
overexpression is oncogenic in fibroblast cell lines and
in xenograft tumour models67. Therefore, deregulation
in TOP gene expression might promote tumorigenesis.
Furthermore, given that most known TOP genes
encode proteins that can upregulate protein synthesis, a
feedback loop might exist in which an increase in S6
phosphorylation results in upregulation in total protein
synthesis in the cell (FIG. 2).
S6 is phosphorylated when quiescent cells reenter the cell cycle in response to mitogens 68.
Phosphorylation of S6 has been observed both
in vivo, during liver regeneration, and in various cell
lines 69,70. Because the level of S6 phosphorylation
increases in proportion to the level of protein synthesis, it has been proposed that S6 could be an important regulator of cell growth, through the regulation
of translation of TOP mRNAs. This has been proven,
in part, in Drosophila, where the inactivation of the
kinase that is responsible for the S6 phosphorylation
(dS6K) results in a decrease in body size and a defect
in cell growth71. There is no data to indicate that this
is due to alterations in translation of TOP genes.
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RAPAMYCIN
Rapamycin is a macrolide with
immunosuppressant properties
and tumour-inhibitory effects.
To exert its effect, rapamycin
binds an immunophilin FK506-binding protein 12, and this
complex inhibits the cellular
target of rapamycin, mTOR.
NATURE REVIEWS | C ANCER
In mammals, the regulation of S6 phosphorylation is
more complex. This increased complexity is due to the
presence of another kinase, S6K2, which is highly
homologous to S6K1 and encodes a protein with redundant function72. Despite the presence of this homologue,
S6 is not phosphorylated in S6K1−/− embryonic stem
cells73. Furthermore, serum stimulation of starved wildtype cells causes increased amounts of the mRNA for
the TOP gene EEF1A1 to associate with the polysome
fraction, whereas this did not occur in S6K1−/− cells.
Overexpression of a dominant-negative form of S6K1
also suppressed the mitogen-induced translation of
chimeric transcripts that contained the 5′ TOP sequence
at the 5′ UTR65. In addition, RAPAMYCIN, a macrolide that
inhibits cellular proliferation by inhibiting the mammalian target of rapamycin (mTOR), reduces S6K1
activity and inhibits the upregulation of TOP mRNAs
after mitogen stimulation65,73 (FIG. 2).
A recent study has challenged these findings, and
has proposed that TOP gene expression is dependent
on the phosphotidyl inositol 3-kinase (PI3K) signalling pathway, but is independent of S6K1 activity74.
Further experiments need to be performed to reconcile these data — for example, by studying cells or mice
that carry gene disruptions in both S6K1 and S6K2
kinases. Importantly, the activity of S6K is markedly
upregulated in tumours that carry mutations in PTEN
and tuberous sclerosis 1/2 (TSC1/2), and therapeutic
agents that disrupt S6K activity were found to slow
tumour growth in mice and in humans (FIG. 2).
The upregulation of r-proteins in cancer cells fits
nicely with their involvement in ribosome production.
Similar to the increase in Pol I transcriptional activity
that results in an increase in rRNA synthesis, r-proteins
could also regulate the number of functional ribosomes
in the cell. In both cases, cells that contain more ribosomes would have an increased translation rate, which
would promote transformation. Although many studies have shown that several r-proteins are overexpressed
by cancer cells, no experiments have been performed to
determine the consequences of this overexpression on
ribosome production and translation.
It is clear that deletion of a single r-protein has a
direct effect on cellular growth in many model organisms, but the consequences of r-protein overexpression
are not as well characterized. In one study, overexpression of the r-protein S3a was able to induce transformation of NIH 3T3 cells and induce formation of tumours
in nude mice75. The ability of S3a to induce transformation is dependent on its role in suppressing programmed cell death, and therefore S3a might cause an
upregulation of anti-apoptotic proteins. However, in
these studies, the effects of S3a on protein translation
have not been investigated. Furthermore, it is difficult to
envision how S3a could specifically induce translation
of anti-apoptotic proteins, so extra-ribosomal functions
have been attributed to S3a by default. The advent of
proteomics will help to identify proteins that are
affected by r-protein overexpression.
It is possible that r-protein overexpression is simply
the result of rapid cellular proliferation. Cancer cells,
which show an increase in cell-cycle progression, might
require more ribosomal components to sustain increased
proliferation. In this case, upregulation of r-proteins
could be an indirect consequence of transformation that
simply maintains the increased proliferation of cancer
cells. However, this has not been proven and it is an
unlikely scenario, given that no data exist to correlate
tumorigenic cells that possess high proliferative rates
with overexpression of specific r-proteins.
Certain r-proteins could also have additional functions. For example, it has been shown that the r-protein
S3 also functions as a DNA-repair enzyme — ultra-violet endonuclease III — that cleaves DNA in response to
ultraviolet irradiation76. Although the extra-ribosomal
function of these proteins might be important for some
aspects of cancer development, the fact that there are no
specific r-proteins associated with specific tumour types
makes this hypothesis unlikely. This issue becomes particularly difficult to address when several different combinations of r-proteins are deregulated in cancer cells.
Ultimately, given the overwhelming number and combination of r-proteins that are overexpressed in a wide
array of cancer types, it is highly unlikely that all of their
effects will be attributed to extra-ribosomal function.
MYC and PTEN
Both the proto-oncogene product MYC and the
tumour suppressor PTEN have been shown to
directly regulate ribosome biogenesis through the
transcription of r-proteins and the regulation of S6K
activity, respectively (FIGS 2,3). c-MYC encodes a transcription factor that is deregulated through genomic
aberrations in a wide array of B-cell-specific malignancies and several other types of neoplasias77. How
is MYC function linked to ribosome biogenesis? This
question has been answered by the finding that r-proteins, as well as factors involved in ribosome assembly, are MYC gene targets78–80 (FIG. 3). Transgenic mice
that constitutively expressed c-MYC under the control of the immunoglobulin heavy-chain enhancer
show increases in cell size. This effect on cell size is
prevalent during all stages of B-cell differentiation
and is independent of the cell cycle, as cells in G0/G1
or G2 are bigger in size compared with the control
cells81. As discussed above, overexpression of a single
r-protein has not been conclusively shown to affect
protein synthesis. However, MYC-overexpressing cells
upregulate r-proteins, resulting in an increase in cell
size and in S35 incorporation. MYC can therefore
regulate total protein synthesis in the cell.
Overexpression of human c-MYC in the liver resulted
in hepatocyte enlargement82. This increase in size
occurred in the absence of cell proliferation and was
associated with enlarged nucleoli. Expression of several
r-proteins and nucleolar proteins that are involved in
ribosome biogenesis were also increased, with respect to
control cells. These findings are in agreement with results
from SERIAL ANALYSIS OF GENE EXPRESSION (SAGE) studies in
neuroblastoma cell lines. In these studies, these cells were
transfected with another member of the MYC oncogene
family, NMYC 79. Most genes upregulated in these cells
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SERIAL ANALYSIS OF GENE
EXPRESSION
(SAGE). A technique that is used
to identify and quantitate genes
that are differentially expressed
between two different samples.
SAGE libraries are generated by
adding a short nucleotide
sequence tag (10 base pairs) to
cDNAs from different RNA
samples that are being analysed.
The abundance of a given tag
within the SAGE library
corresponds to the expression
level of the corresponding gene,
which can easily be identified by
sequencing directly from the
transcript tag.
were r-proteins associated with the large and small ribosomal subunits (FIG. 3). In addition, analysis of primary
neuroblastoma samples with an amplification of endogenous NMYC revealed an overexpression of genes that
encode components of the protein synthesis machinery79.
A second group of genes upregulated in NMYCtransfected cells consist of nucleolar proteins such as
B23 (nucleophosmin or NPM) and nucleolin78–80,82.
These two gene products were also found to be upregulated in c-MYC-overexpressing hepatocytes. These
nucleolar proteins function in the processing of rRNA
precursors, and have been implicated in the regulation
of ribosome assembly or the nucleo-cytoplasmatic
transport of mature ribosomal subunits. Nine translation initiation and elongation factors were also induced
in these cells. These results support a role for MYC-family members as key regulators of ribosome biogenesis
and translation control. The ability of MYC to regulate
the transcription of several components of the protein
synthesis machinery has been validated in several different cell types and corresponds with both MYC loss and
gain of function. Furthermore, the ability of MYC to
regulate the expression of this class of genes is dependent on its ability to regulate transcription. This has
been shown in cells that overexpress MAD1, a transcriptional repressor of MYC target genes. Cell growth is
inhibited in these cells, and genes that are involved in
ribosome biogenesis and translation control are
repressed83. It remains to be determined which of these
genes have MYC DNA-binding sites within their promoters and which, by contrast, are in pathways indirectly
controlled by MYC.
MYC
Ribosomal proteins: L3, L15, L39, L37, S2, S3a, S6, etc
Nucleolar proteins: B23, nucleolin, fibrillarin, etc
Translation factors: elF-4A, eIF-4E, elF3, EEF1α1, etc
Ribosome biogenesis and
translation control
Figure 3 | MYC regulates the expression of proteins
involved in ribosome biogenesis and control of
translation. The proto-oncogene MYC encodes a
transcription factor that is deregulated through genomic
aberrations in a wide array of B-cell-specific malignancies and
several other types of neoplasias. Most genes that are found
to be upregulated in MYC-overexpressing cells are ribosomal
proteins associated with the large and small ribosomal
subunits. In addition, several nucleolar proteins are found
upregulated, including B23, which is overexpressed in a wide
array of tumours and might have direct consequences for
tumour progression that is initiated by MYC. The translation
factor eIF-4E is also itself a proto-oncogene and is one of
many translation factors found upregulated in MYCoverexpressing cells. The ability of MYC to regulate the
expression of these classes of mRNAs corresponds to an
increase in protein synthesis observed in MYC-overexpressing
cells. Regulation of protein synthesis could therefore be an
important mechanism by which MYC regulates cell growth
and initiates tumorigenesis.
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It will be essential to determine which of the MYC
target genes involved in regulation of protein synthesis
also promote transformation, and whether an increase
in ribosome biogenesis and translation of specific
mRNAs directly mediates MYC’s oncogenic effects.
Overexpression of MYC has been shown to increase cell
size — possibly to accommodate the expanded proteinsynthesis capacity of these cells81–84. Whether the expansion in cell size per se has an affect on the susceptibility
of these cells to undergo transformation remains
unknown. Although beyond the scope of this review,
much research is currently focused on whether the ability of proto-oncogenes, such as MYC, to regulate cell size
would make these cells more susceptible to acquire
secondary mutations involved in cancer progression
(see REFS 85,86 for reviews).
The tumour suppressor PTEN87,88 has also been
shown to regulate cell size in association with its ability
to regulate ribosome biogenesis89,90. PTEN is a phosphatase that downregulates the PI3K pathway by
dephosphorylating the lipid phosphatidylinositol-3,4,
5-trisphosphate to phosphatidylinositol-4,5-bisphosphate (REFS 91,92). PI3K signalling leads to an increase in
S6K activity, concomitant with hyperphosphorylation of
the S6 ribosomal protein86 (FIG. 2). These events are positively regulated by the kinase mTOR (a target of
rapamycin), although it is unclear whether mTOR
directly phosphorylates S6K in physiological conditions93–96. PTEN acts upstream mTOR and has a negative
effect on the phosphorylation of S6 (REFS 97,98). In fact,
PTEN-null embryonic stem cells show an increase in
S6K activity and display constitutive phosphorylation of
S6. A linear model has been proposed for S6 activation,
whereby AKT signals to S6K through the activation of
mTOR, and PTEN negatively regulates this process99,100.
Additionally, it was shown that the plekstrin homology
(PH)-domain containing protein, phosphoinositidedependent kinase (PDK1), which phosphorylates AKT
at the cell membrane, also directly phosphorylates S6K
in an AKT-independent manner101 (FIG. 2).
PTEN is mutated in many different types of tumour,
but how important is its ability to regulate mTOR activity in coordinating tumour suppression? Recent reports
have highlighted the importance of mTOR in PI3Kdependent oncogenesis. Transformation by PI3K or
AKT directly correlates with mTOR activation and activation of its downstream target S6K in primary cultures
of chicken embryo fibroblasts102. Rapamycin, which
inhibits mTOR, prevents induction of transformed cell
foci by PI3K and AKT, but does not have an effect on
cells transformed with other oncogenes such as v-Jun or
v-Src102. Pten+/− mice are more prone to developing
tumours of different histological origins, which are
associated in some cases with PTEN loss of heterozygosity 87,88,103,104. Data from different reports indicate loss of
PTEN function in tumours with increases in AKT and
S6K kinase activity. Pharmacological inactivation of
mTOR by rapamycin reduces tumour-cell proliferation
and tumour size in Pten+/− mice98. This tumour regression is associated with the downregulation of S6K
activity to wild-type levels. In tumours derived from
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REVIEWS
murine Pten−/− embryonic stem cells, or in human
tumour cell lines in which PTEN is lost, rapamycin
treatment led to a reduction in cell size97. The drug has
no effect on tumour cells without PTEN mutations.
This supports the role of PTEN in growth regulation
through S6K activation. The mTOR pathway and the
downstream effector S6K seem to be involved in
tumorigenesis, and rapamycin and its analogues might
therefore be developed as chemotherapeutic agents.
Another tumour suppressor that was recently shown
to regulate S6K activity is the heterodimeric complex
TSC1/2, which is mutated in patients with tuberous
sclerosis105. This disease is associated with cancer susceptibility, including haemartoma growth. The TSC1/2
complex in Drosophila, as well as in mammalian cells,
acts as an inhibitor of S6K activity106–110 (FIG. 2). TSC2
mutations are also associated with tumour development
in lymphangioleiomyomatosis (LAM) in primary
tumours, as well as in cell lines derived from these
tumours, which display constitutive activation of S6K.
The hyperphosphorylation of S6 is associated with an
increase in cell proliferation111.
So, does the TSC1/2 complex directly inactivate
S6K activity, or does regulation of S6 phosphorylation
by TSC1/2 depend on other components of PI3K
pathway, such as mTOR? Although there is more data
to support the model that the TSC1/2 complex inhibits
mTOR106–109, and this in turn results in inactivation of
S6K activity, other data place mTOR in a parallel pathway112. If the former model is correct, then PTEN and
TSC1/2 could cooperate to repress cellular growth and
proliferation, and concomitant deregulation of both of
these tumour suppressors might have an additive
function during tumorigenesis. Regardless of the precise mechanisms by which S6K activation is perturbed
in tumours, the fact remains that mutations in PTEN
and TSC1/2, as well as amplification of the AKT gene,
all have a common downstream r-protein target,
which is S6.
Unfortunately, although emphasis has been placed
on how TSC1/2 and PTEN regulate SK6, the downstream effects of S6K hyperactivation in cancer remain
less characterized. The fact that S6 is phosphorylated
makes it a highly regulated r-protein, and is constitutively activated in several tumours. Whether this results
in upregulation of TOP gene expression or an increase
in total protein synthesis needs to be determined. It will
also be important to establish whether rapamycin treatment also has an effect on the initiation factor of translation, eIF4E, whose activity is regulated by mTOR99.
Because eIF4E has been shown to act as a proto-oncogene113–115, it might also be involved in deregulated protein synthesis that is elicited by the loss of PTEN and
TSC1/2 tumour suppressors.
Further experimental evidence is required to link
the activity of ribosomal proteins such as S6 to translation defects and cancer. Crosses between Pten-mutant
mice and mice deficient in components of the mTOR
signal-transduction pathway will clarify to which extent
activation of mTOR is a prerequisite for neoplastic
transformation. In addition, it will be worthwhile to
NATURE REVIEWS | C ANCER
investigate in PTEN-null cells whether protein translation is impaired as a result of constitutive activation of
the mTOR pathway. Given that S6K is involved in the
control of TOP gene translation, it will be particularly
informative to study the expression of this subset of
proteins in Pten-mutant mice and cells.
Cancer and ribosome biogenesis
Oncogenes and tumour suppressors can contribute to
cancer progression through alterations in ribosome
biogenesis. Genetics studies have shown that components of the protein synthesis machinery can be
directly mutated and are responsible for disease
syndromes that are characterized by an increased susceptibility to cancer. Could disruption of ribosome
function be the first oncogenic event in a multi-hit
tumour-formation process?
Dyskeratosis congenita. DKC1 encodes a putative
pseudouridine synthase, which mediates post-transcriptional modification of ribosomal RNA (rRNA)
through the site-specific conversion of uridine (U) to
pseudouridine (ψ)116,117 (BOX 1). Mutations in DKC1 are
associated with dyskeratosis congenita (DC), a disease
that is characterized by features of premature ageing,
including bone-marrow failure and hyperkeratosis of
the skin2,118. Importantly, patients with DC show an
increased susceptibility to tumour formation. In yeast
and fly, mutations in DKC1 result in impaired rRNA
modifications associated with a reduction in ribosome
biogenesis119,120. It is therefore tempting to speculate
that the molecular pathogenesis of DC and the
tumour susceptibility observed in DC patients result
from defects in ribosome modification. However, the
possibility of a direct involvement in ribosome biogenesis as a cause of the disease has remained controversial, given that DKC1 is also associated with the RNA
component of the telomerase complex121. Whether
ribosome dysfunction is the underlying cause of DC
pathogenesis has been tested in animal models3. The
mouse represents an ideal model organism for
addressing whether DKC1 mutations result in defects
in rRNA modification, telomere maintenance, or both,
as defects in telomerase function leading to telomere
attrition would only be manifest in late generations122.
Hypomorphic Dkc1 mutant (Dkc1m) mice3 recapitulate, in first generations, the clinical features of DC.
Dkc1m cells from early-generation mice are impaired in
rRNA pseudouridylation before disease onset. These
results indicate that deregulated ribosome function
is involved in the initiation of DC. More than 50%
of Dkc1-mutant mice develop tumours during their
lifespan, indicating that Dkc1 is an important tumoursuppressor gene. Dkc1m mice also show defects in
rRNA processing (BOX 1), and cells from these mice are
hypersensitive to translation inhibitors, which are used
to quantify ribosome function.
How can alterations in rRNA modification promote
neoplastic transformation? DKC1 has been shown to
mediate pseudouridylation. Although the specific
function of the pseudouridine rRNA modification
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remains to be determined, it has been shown that modification sites are strategically placed within specific
domains of the ribosome123. For example, modifications
have been found in regions of the ribosome that are
important for tRNA and mRNA binding. The reduction
in modified uridine residues in the ribosome could result
in impaired translation of specific mRNAs that are
important for cellular transformation.
For example, genes encoding tumour suppressors
such as p27 and p58 have been shown to contain
5′ leader sequences known as internal ribosome entry
sites (IRES), which facilitate interaction of the ribosome with mRNA to promote translation124,125.
Translation of these types of mRNAs requires more
direct contacts with the ribosome and would therefore
be more sensitive to alterations in ribosome folding.
Given that co-crystallization of Escherichia coli DKC1
with RNA revealed a potential role for pseudouridine
synthases as RNA chaperones that are important for
proper ribosome folding126, DKC1-mutant cells might
possess misfolded ribosomes. These mutant ribosomes
could be impaired in the translation of mRNAs with
IRES sequences124,125,127. The identification of genes
that are deregulated in Dkc-mutant mice will improve
our understanding of how pseudouridylation might
affect ribosome function and contribute to cancer.
Diamond–Blackfan anaemia. Another cancer susceptibility syndrome that is directly associated with
defects in ribosome biogenesis is Diamond–Blackfan
anaemia, which is characterized by anaemia and an
increased susceptibility to haematopoietic malignancies128. This disease has been associated with mutations
in the r-protein S19, providing direct evidence that loss
of r-protein function can cause cancer4. Although the
molecular mechanisms by which a reduction in the
expression or activity of S19 causes the cancer predisposition are unknown, several features of the disease
state indicate that patients are impaired in ribosome
function. As in Drosophila, in which mutations in a
single r-protein result in the minute phenotype,
patients with Diamond–Blackfan anaemia have
hypotrophy at birth and show severe growth retardations128. As r-proteins are highly conserved between
Drosophila and humans, is it likely that defects in
human r-proteins would result in overall ribosomal
dysfunction, and that this could lead to growth defects.
However, as stated above, the possibility for an extraribosomal function for S19 cannot be excluded.
Because r-protein expression is upregulated in
several types of cancer, it remains to be determined
how the loss of an r-protein would contribute to neoplasm. Given the specificity in the pathological features that are observed in Diamond–Blackfan
anaemia patients, it has yet to be seen whether S19
can regulate translation in a qualitative manner, as
proposed for the S6 r-protein, which facilitates translation of 5′ TOP genes. Therefore, downregulation in
translation of specific mRNA, such as those encoding
tumour suppressors, could be a possible mechanism
for the loss of a r-protein. Creation of animal models
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for Diamond–Blackfan anaemia and primary cells that
lack S19 will be useful in determining the mechanism
by which ribosome biogenesis promotes cancer.
Future directions
Several pathways that lead to increased ribosome biogenesis have been associated with the transformation
process (BOX 2). Alterations can occur either at the level
of rRNA transcription, modification or assembly of the
ribosome. Evidence has clearly shown that several
tumour suppressors and oncogenes regulate ribosome
function, but determining the precise effects of these
interactions will require further investigation. We have
loosely used the term ‘increased total translation’
throughout the review as an obvious potential outcome
for increased ribosome production. Although this
might occur in tumours that show increases in rRNA
synthesis, how can increased translation predispose cells
to neoplasm? At the heart of this question is the debate
about whether increased cellular growth or proliferation
brought about by elevated proteins synthesis is a means
to cancer progression, or whether it actually serves as a
bona fide ‘hit’ which itself is necessary and sufficient for
cancer initiation. Several possibilities exist, each of
which remain highly speculative (BOX 2).
One consequence of an increase in translation of cellular mRNAs would be the increased production of certain proteins that normally show low or moderate levels
of expression. Expression of most oncogenes is tightly
regulated to prevent deleterious effects on cell growth.
One mechanism by which cells regulate oncogene
expression involves the long, highly structured 5′ UTR
that is present in these mRNAs129. Initiation factors are
believed to unwind the secondary structure of 5′ UTRs
and recruit ribosomes to these sites. This process limits
the expression rate of these proteins. It has been proposed that initiation factors such as eIF4E could be rate
limiting in the cell, and that their upregulation might
result in increased cancer susceptibility through
enhanced translation of mRNAs with a highly structured 5′ UTR114,115. An upregulation in ribosome production could facilitate the translation of these mRNAs
by increasing the rate of formation of the initiation
complex. It is worth noting that the overexpression of
proto-oncogenes such as MYC results not only in an
upregulation in ribosomal proteins but also in initiation
factors for translation (FIG. 3). Therefore, an increase in
translation, mediated by a rise in ribosome production
and initiation factors, could actually promote translation of proteins involved in cellular growth control that
are the least abundantly expressed in the cell and often
encode proto-oncogenes.
Specificity in translation of certain mRNAs could
also be achieved by an upregulation of certain r-proteins. As discussed above, phosphorylation of S6
results in an increase of specific target mRNAs that
contain a terminal oligopyrimidine tract in their 5′
UTR. S6 is constitutively phosphorylated in many
types of cancer cell, and the activity of tumour suppressors that is important for the phosphorylation of
this ribosomal protein is also deregulated. However,
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Box 2 | Different steps in the pathway to ribosome production that can lead to cancer, when deregulated
The regulation of ribosome biogenesis is a highly coordinated
process that leads to accurate initiation and regulation of
protein synthesis. There are three steps in this process, which,
when deregulated, can contribute to increased tumorigenesis.
The first step in ribosome production requires the synthesis of
the 45S rRNA precursor. The transcription of this rRNA gene
is negatively regulated by tumour suppressors such as p53 and
retinoblastoma (RB) and augmented on mitogenic stimuli by
several kinases that phosphorylate components of the
transcription complex that are responsible for 45S synthesis.
The accurate regulation of rRNA synthesis can be lost in
cancer cells through inactivating mutations in tumour
suppressors or upregulation of these kinases.
Another step in ribosome biogenesis that maintains
accurate cellular function involves the modification of rRNA.
One type of modification, which is catalysed by an enzyme
known as dyskerin, converts uridine into pseudouridine (Ψ).
Mutations in the gene encoding dyskerin, DKC1, result in
increased tumour susceptibility. Animal models that have lost
DKC1 function show a marked increase in tumour incidence
associated with a decrease in rRNA processing.
Ribosome assembly, which involves the association of rRNA
with more than 70 ribosomal proteins (made in the
cytoplasm), is another important step. The rRNA and proteins
are assembled into the large subunit (60S) and small subunit
(40S) of the ribosome. An increase in ribosomal protein
production and activity has been observed in many cancer
types. Mutations in ribosomal proteins such as S19 have also
been associated with a human syndrome that is characterized
by increased tumour susceptibility.
Each of the three highlighted steps (blue boxes) might have
deleterious effects on the cell that could contribute to tumour
initiation or cancer progression, or both, through aberrant
regulation of protein synthesis. This can be manifested either
by an increase in ribosome production, thereby leading to an
upregulation in total translation, or by alterations in
translation of specific mRNAs, which are involved in the
regulation of cell proliferation. Furthermore, when key
checkpoints in the cell that are important in coordinating
ribosome production with accurate cell-cycle progression are
lost,‘nucleolar stress’ can result, and subsequently
unrestrained cellular proliferation occurs. The advent of
proteomics will aid in identifying protein targets that are
deregulated as a result of perturbations in these pathways.
Increase in rRNA synthesis
by inactivating mutations in
tumour suppressors
rRNA gene
45S rRNA
precursor transcription
Nucleus
ψ
5′
Modification
ψ
ψ
3′
CH3
CH3
Nucleolus
Processing and
modifications by
snoRNPs and RNase
CH3
ψ
ψ
CH3
28S
CH3
Ribosome
assembly
ψ
18S
5,8S
5S
CH3
Ribosomal
proteins made
in cytoplasm
Large subunit
(60 S)
Small subunit
(40 S)
Cytoplasm
Mutations and
overexpression
of r-proteins
it remains unclear whether the modification of S6
contributes at the level of translation of its target
mRNAs towards tumorigenesis. Modification of
other r-proteins following mitogen activation could
similarly regulate the translation of specific mRNAs.
For example, r-proteins were recently shown to be
ubiquitylated, and this modification corresponds
with their selective destruction during a specific window of the cell cycle 130. These and other types of
modifications could provide the ribosome with
specificity for certain messengers, or enable cell-cycle
control of ribosome function to take place. The precise consequence of r-protein overexpression, and
whether these mechanisms promote cellular transformation, remains to be determined.
NATURE REVIEWS | C ANCER
Alterations in the
modification of rRNA
(DKC1)
• Increased total translation
• Altered translation of specific mRNAs
• Nucleolar stress and deregulated
cell-cycle progression
Although ribosome biogenesis is tightly linked to regulation of translation, and cells that overexpress MYC
show an increase in total translation, other possibilities
exist for how alterations in ribosome biogenesis could
lead to cancer. This is highlighted by findings during the
course of analysing the S6−/− mice62. Although loss of this
r-protein results in decreased ribosome biogenesis, total
cellular translation is relatively unaffected. However, following challenge, the S6-null cells show a defect in proliferation. This defect occurred concomitantly with a
decrease in cyclin E at the mRNA level, which is important for the G1-to-S transition. How could alterations in
ribosome biogenesis per se affect the cell cycle?
Growth and proliferation are associated with changes
in the rate of ribosome production, and ribosome
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biogenesis can serve as a sensor for cells to bypass
important checkpoints during the cell cycle. In transformed cells that show increased ribosome production,
the cell cycle can be altered as a consequence of alterations in ribosome biogenesis. This tight feedback
between the ribosome and the cell cycle could function
to maintain cellular homeostasis. Therefore, an increase
in ribosome amount, irrespective of any consequent
affects on protein translation, could contribute to the
transformation process.
Unfortunately, the proteins that are responsible for
mediating cross-talk between the ribosome and the
cell cycle remain largely unknown. For example, the
mechanism by which S6-null cells, which contain
fewer ribosomes than cells that express S6, show a
selective downregulation of cyclin E mRNA at the
transcript level is unknown. Interestingly, a novel
nucleolar protein involved in rRNA processing known
as BOP1 (REF. 131) has been shown to cooperate with
p53 in regulating the G1-to-S transition132. The ability
of a dominant-negative form of Bop1 to induce cellcycle arrest was abrogated in p53-null cells, but rRNA
processing was still impaired. These findings indicate
that the p53 pathway could link ribosome biogenesis
and the cell cycle. Alterations in ribosome biogenesis in
1.
Gani, R. The nucleoli of cultured human lymphocytes. I.
Nucleolar morphology in relation to transformation and the
DNA cycle. Exp. Cell Res. 97, 249–258 (1976).
2. Heiss, N. S. et al. X-linked dyskeratosis congenita is
caused by mutations in a highly conserved gene with
putative nucleolar functions. Nature Genet. 19, 32–38
(1998).
3. Ruggero, D. et al. Dyskeratosis congenita and cancer in
mice deficient in ribosomal RNA modification. Science 299,
259–262 (2003).
In this study, animal models for dyskeratosis
congenita were generated, which are hypomorphic
mutants for the pseudouridine synthase, DKC1. These
mice are faithful models of the disease and show
marked cancer susceptibility associated with an
impairment in ribosome biogenesis.
4. Draptchinskaia, N. et al. The gene encoding ribosomal
protein S19 is mutated in Diamond-Blackfan anaemia.
Nature Genet. 21, 169–175 (1999).
Mutations in the ribosomal protein S19 were found to
be associated with a disease characterized by an
increased susceptibility to cancer.
5. Pardee, A. B. G1 events and regulation of cell proliferation.
Science 246, 603–608 (1989).
6. Pyronnet, S. & Sonenberg, N. Cell-cycle-dependent
translational control. Curr. Opin. Genet. Dev. 11, 13–18
(2001).
7. Grummt, I. Regulation of mammalian ribosomal gene
transcription by RNA polymerase I. Prog. Nucleic Acid Res.
Mol. Biol. 62, 109–154 (1999).
8. Grummt, I., Smith, V. A. & Grummt, F. Amino acid starvation
affects the initiation frequency of nucleolar RNA polymerase.
Cell 7, 439–445 (1976).
9. Kief, D. R. & Warner, J. R. Coordinate control of syntheses of
ribosomal ribonucleic acid and ribosomal proteins during
nutritional shift-up in Saccharomyces cerevisiae. Mol. Cell.
Biol. 1, 1007–1015 (1981).
10. Klein, J. & Grummt, I. Cell cycle-dependent regulation of
RNA polymerase I transcription: the nucleolar transcription
factor UBF is inactive in mitosis and early G1. Proc. Natl
Acad. Sci. USA 96, 6096–6101 (1999).
An important paper showing that UBF activity is
regulated in a cell-cycle-dependent manner and that
fluctuations in rRNA synthesis during the cell cycle
are controlled by UBF phosphorylation.
11. Cavanaugh, A. H. et al. Activity of RNA polymerase I
transcription factor UBF blocked by Rb gene product.
Nature 9, 177–180 (1995).
190
| MARCH 2003 | VOLUME 3
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
cancer cells might result in a general ‘nucleolar stress’,
leading to alterations in cell-cycle progression. In this
model, the rate or efficiency of ribosome production in
the cell could serve as a signal by which cells could
negatively or positively regulate cell-cycle progression
independently from its effect on protein synthesis.
A significant challenge in the future will be to
understand the relative contribution of proteins
directly involved in ribosome biogenesis and the control of translation, as these could be important to the
molecular aetiology of specific tumour types, as well
as their precise effects within the context of a multistep pathway that leads to cancer. This avenue
of research will be greatly benefited by the advent of
proteomics, which will enable the identification of
proteins directly deregulated as a result of alterations
in ribosome biogenesis.
The finding that components of the translation
machinery are overexpressed or deregulated in cancer
cells could lead to the discovery of new therapeutic
agents, designated to target factors that control translation. A compelling example in this respect is represented by rapamycin, which specifically inactivates
mTOR and has been recently shown to act as a
powerful tumour-suppressive drug in clinical trials133.
A landmark paper which shows that the tumoursuppressor gene Rb directly represses transcription
of rRNA through inhibition of UBF function. There is
an accumulation of Rb in the nucleolus of
differentiated cells, indicating that Rb might exert
some of its growth-suppressive properties by
regulating rRNA synthesis.
Beckmann, H., Chen, J. L., O’Brien, T. & Tjian, R.
Coactivator and promoter-selective properties of RNA
polymerase I TAFs. Science 270, 1506–1509 (1995).
Brandenburger, Y., Jenkins, A., Autelitano, D. J. &
Hannan, R. D. Increased expression of UBF is a critical
determinant for rRNA synthesis and hypertrophic growth of
cardiac myocytes. FASEB J. 15, 2051–2053 (2001).
Bell, S. P., Learned, R. M., Jantzen, H. M. & Tjian, R.
Functional cooperativity between transcription factors UBF1
and SL1 mediates human ribosomal RNA synthesis.
Science 241, 1192–1197 (1988).
antzen, H. M., Admon, A., Bell, S. P. & Tjian, R. Nucleolar
transcription factor hUBF contains a DNA-binding motif with
homology to HMG proteins. Nature 344, 830–836 (1990).
Read, C. et al. High resolution studies of the Xenopus laevis
ribosomal gene promoter in vivo and in vitro. J. Biol. Chem.
267, 10961–10967 (1992).
Voit, R., Hoffmann, M. & Grummt, I. Phosphorylation by
G1-specific cdk-cyclin complexes activates the nucleolar
transcription factor UBF. EMBO J. 18, 1891–1899 (1999).
Kihm, A. J., Hershey, J. C., Haystead, T. A., Madsen, C. S. &
Owens, G. K. Phosphorylation of the rRNA transcription
factor upstream binding factor promotes its association with
TATA binding protein. Proc. Natl Acad. Sci. USA 95,
14816–14820 (1998).
Tuan, J. C., Zhai, W. & Comai, L. Recruitment of TATAbinding protein-TAFI complex SL1 to the human ribosomal
DNA promoter is mediated by the carboxy-terminal
activation domain of upstream binding factor (UBF) and is
regulated by UBF phosphorylation. Mol. Cell. Biol. 19,
2872–2879 (1999).
Voit, R. & Grummt, I. Phosphorylation of UBF at serine 388
is required for interaction with RNA polymerase I and
activation of rDNA transcription. Proc. Natl Acad. Sci. USA
98, 13631–13636 (2001).
Stefanovsky, V. Y. et al. An immediate response of ribosomal
transcription to growth factor stimulation in mammals is
mediated by ERK phosphorylation of UBF. Mol. Cell 8,
1063–1073 (2001).
The study shows that the activation of the ERK1/2
MAP kinases by growth factors leads to rRNA
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
transcription through a mechanism that is dependent
on UBF phosphorylation. This study provides a link
between growth-factor signalling and increased
ribosome production.
O’Mahony, D. J., Xie, W. Q., Smith, S. D., Singer, H. A. &
Rothblum, L. I. Differential phosphorylation and localization
of the transcription factor UBF in vivo in response to serum
deprivation. In vitro dephosphorylation of UBF reduces its
transactivation properties. J. Biol. Chem. 267, 35–38 (1992).
Voit, R. et al.The nucleolar transcription factor mUBF is
phosphorylated by casein kinase II in the C-terminal
hyperacidic tail which is essential for transactivation.
EMBO J. 11, 2211–2218 (1992).
Voit, R., Kuhn, A., Sander, E. E. & Grummt, I. Activation of
mammalian ribosomal gene transcription requires
phosphorylation of the nucleolar transcription factor UBF.
Nucleic Acids Res. 23, 2593–2599 (1995).
Wang, T. C. et al. Mammary hyperplasia and carcinoma in
MMTV-cyclin D1 transgenic mice. Nature 369, 669–671
(1994).
Bortner, D. M. & Rosenberg, M. P. Induction of mammary
gland hyperplasia and carcinomas in transgenic mice
expressing human cyclin E. Mol. Cell. Biol. 17, 453–459
(1997).
Navolanic, P. M., Steelman, L. S. & McCubrey, J. A. EGFR
family signaling and its association with breast cancer
development and resistance to chemotherapy. Int. J. Oncol.
22, 237–252 (2003).
Cavanaugh, A. H. et al. Rrn3 phosphorylation is a regulatory
checkpoint for ribosome biogenesis. J. Biol. Chem. 277,
27423–27432 (2002).
Yuan, X., Zhao, J., Zentgraf, H., Hoffmann-Rohrer, U. &
Grummt, I. Multiple interactions between RNA polymerase I,
TIF-IA and TAFI subunits regulate preinitiation complex
assembly at the ribosomal gene promoter. EMBO Rep. 3,
1082–1087 (2002).
Classon, M. & Harlow, E. The retinoblastoma tumour
suppressor in development and cancer. Nature Rev. Cancer
2, 910–917 (2002).
Voit, R., Schafer, K. & Grummt, I. Mechanism of repression
of RNA polymerase I transcription by the retinoblastoma
protein. Mol. Cell. Biol. 17, 4230–4237 (1997).
Hannan, K. M. et al. Rb and p130 regulate RNA polymerase
I transcription: Rb disrupts the interaction between UBF and
SL-1. Oncogene 19, 4988–4999 (2000).
Ciarmatori, S. et al. Overlapping functions of the pRb family
in the regulation of rRNA synthesis. Mol. Cell. Biol. 21,
5806–5814 (2001).
www.nature.com/reviews/cancer
REVIEWS
34. Hannan, K. M. et al. RNA polymerase I transcription in
confluent cells: Rb downregulates rDNA transcription during
confluence-induced cell cycle arrest. Oncogene 19,
3487–3497 (2000).
35. Zhai, W. & Comai, L. Repression of RNA polymerase I
transcription by the tumor suppressor p53. Mol. Cell. Biol.
20, 5930–5938 (2000).
Shows that the tumour suppressor p53 can directly
regulate rRNA synthesis through its interaction with
UBF.
36. Cordon-Cardo, C. et al. Cooperative effects of p53 and pRB
alterations in primary superficial bladder tumors. Cancer
Res. 57, 1217–1221 (1997).
37. White, R. J., Trouche, D., Martin, K., Jackson, S. P. &
Kouzarides, T. Repression of RNA polymerase III transcription
by the retinoblastoma protein. Nature 382, 88–90 (1996).
38. Sutcliffe, J. E., Brown, T. R., Allison, S. J., Scott, P. H. &
White, R. J. Retinoblastoma protein disrupts interactions
required for RNA polymerase III transcription. Mol. Cell. Biol.
20, 9192–9202 (2000).
39. Cairns, C. A. & White, R. J. p53 is a general repressor of RNA
polymerase III transcription. EMBO J. 17, 3112–3123 (1998).
40. Kaye, F. J., Kratzke, R. A., Gerster, J. L. & Horowitz, J. M.
A single amino acid substitution results in a retinoblastoma
protein defective in phosphorylation and oncoprotein
binding. Proc. Natl Acad. Sci. USA 87, 6922–6926 (1990).
41. Larminie, C. G., Alzuherri, H. M., Cairns, C. A., McLees, A. &
White, R. J. Transcription by RNA polymerases I and III:
a potential link between cell growth, protein synthesis and
the retinoblastoma protein. J. Mol. Med. 76, 94–103 (1998).
42. Johnson, L. F., Williams, J. G., Abelson, H. T., Green, H. &
Penman, S. Changes in RNA in relation to growth of the
fibroblast. III. Posttranscriptional regulation of mRNA
formation in resting and growing cells. Cell 4, 69–75 (1975).
43. Baxter, G. C. & Stanners, C. P. The effect of protein
degradation on cellular growth characteristics. J. Cell.
Physiol. 96, 139–145 (1978).
44. Frank, D. J., Edgar, B. A. & Roth, M. B. The Drosophila
melanogaster gene brain tumor negatively regulates cell
growth and ribosomal RNA synthesis. Development 129,
399–407 (2002).
45. Frank, D. J. & Roth, M. B. ncl-1 is required for the regulation
of cell size and ribosomal RNA synthesis in Caenorhabditis
elegans. J. Cell Biol. 140, 1321–1329 (1998).
46. Fatica, A. & Tollervey, D. Making ribosomes. Curr. Opin. Cell.
Biol. 14, 313–318 (2002).
47. Draper, D. E. & Reynaldo, L. P. RNA binding strategies of
ribosomal proteins. Nucleic Acids Res. 27, 381–388
(1999).
48. Maguire, B. A. & Zimmermann, R. A. The ribosome in focus.
Cell 104, 813–816 (2001).
49. Ramakrishnan, V. Ribosome structure and the mechanism
of translation. Cell 108, 557–572 (2002).
50. Ferrari, S. et al. Noncoordinated expression of S6, S11, and
S14 ribosomal protein genes in leukemic blast cells. Cancer
Res. 50, 5825–5828 (1990).
51. Loging, W. T. & Reisman, D. Elevated expression of
ribosomal protein genes L37, RPP-1, and S2 in the
presence of mutant p53. Cancer Epidemiol. Biomarkers
Prev. 8, 1011–1016 (1999).
52. Kondoh, N. et al. Enhanced expression of S8, L12, L23a,
L27 and L30 ribosomal protein mRNAs in human
hepatocellular carcinoma. Anticancer Res. 21, 2429–2433
(2001).
53. Zhang, L. et al. Gene expression profiles in normal and
cancer cells. Science 276, 1268–1272 (1997).
54. Alon, U. et al. Broad patterns of gene expression revealed
by clustering analysis of tumor and normal colon tissues
probed by oligonucleotide arrays. Proc. Natl Acad. Sci. USA
96, 6745–6750 (1999).
55. Uechi, T., Tanaka, T. & Kenmochi, N. A complete map of the
human ribosomal protein genes: assignment of 80 genes to
the cytogenetic map and implications for human disorders.
Genomics 72, 223–230 (2001).
56. Bassoe, C. F., Bruserud, O., Pryme, I. F. & Vedeler, A.
Ribosomal proteins sustain morphology, function and
phenotype in acute myeloid leukemia blasts. Leuk. Res. 22,
329–339 (1998).
57. Ferrari, S. et al. Abundance of the primary transcript and its
processed product of growth-related genes in normal and
leukemic cells during proliferation and differentiation. Cancer
Res. 52, 11–16 (1992).
58. Warner, J. R. The economics of ribosome biosynthesis in
yeast. Trends Biochem. Sci. 24, 437–440 (1999).
59. Rotenberg, M. O., Moritz, M. & Woolford, J. L. Jr. Depletion
of Saccharomyces cerevisiae ribosomal protein L16 causes
a decrease in 60S ribosomal subunits and formation of halfmer polyribosomes. Genes Dev. 2, 160–172 (1988).
60. Kongsuwan, K. et al. A Drosophila Minute gene encodes a
ribosomal protein. Nature 317, 555–558 (1985).
NATURE REVIEWS | C ANCER
61. Kay, M. A. & Jacobs-Lorena, M. Developmental genetics of
ribosome synthesis in Drosophila. Trends Genet. 3,
347–351 (1987).
62. Volarevic, S. et al. Proliferation, but not growth, blocked by
conditional deletion of 40S ribosomal protein S6. Science
288, 2045–2047 (2000).
63. Terada, N. et al. Rapamycin selectively inhibits translation of
mRNAs encoding elongation factors and ribosomal proteins.
Proc. Natl Acad. Sci. USA 91, 11477–11481 (1994).
64. Jefferies, H. B., Reinhard, C., Kozma, S. C. & Thomas, G.
Rapamycin selectively represses translation of the
‘polypyrimidine tract’ mRNA family. Proc. Natl Acad. Sci.
USA 91, 4441–4445 (1994).
65. Jefferies, H. B. et al. Rapamycin suppresses 5′ TOP mRNA
translation through inhibition of p70s6k. EMBO J. 16,
3693–3704 (1997).
66. Meyuhas, O. & Hornstein, E. in Translational Control of Gene
Expression (eds Sonenberg, N., Hershey, J. W. B. &
Mathews, M. B.) (Cold Spring Harbor Laboratory Press,
New York, 2000).
67. Anand, N. et al. Protein elongation factor EEF1A2 is a
putative oncogene in ovarian cancer. Nature Genet. 31,
301–305 (2002).
68. Thomas, G., Siegmann, M. & Gordon, J. Multiple
phosphorylation of ribosomal protein S6 during transition of
quiescent 3T3 cells into early G1, and cellular
compartmentalization of the phosphate donor. Proc. Natl
Acad. Sci. USA 76, 3952–3956 (1979).
69. Gressner, A. M. & Wool, I. G. The phosphorylation of liver
ribosomal proteins in vivo. Evidence that only a single small
subunit protein (S6) is phosphorylated. J. Biol. Chem. 249,
6917–6925 (1974).
70. Kozma, S. C. Thomas G. p70s6k/p85s6k: mechanism of
activation and role in mitogenesis. Cancer Biol. 5, 255–260
(1994).
71. Montagne, J. et al. Drosophila S6 kinase: a regulator of cell
size. Science 285, 2126–2129 (1999).
72. Shima, H. et al. Disruption of the p70(s6k)/p85(s6k) gene
reveals a small mouse phenotype and a new functional S6
kinase. EMBO J. 17, 6649–6659 (1998).
73. Kawasome, H. et al. Targeted disruption of p70(s6k) defines
its role in protein synthesis and rapamycin sensitivity. Proc.
Natl Acad. Sci. USA 95, 5033–5038 (1998).
74. Stolovich, M. et al. Transduction of growth or mitogenic
signals into translational activation of TOP mRNAs is fully
reliant on the phosphatidylinositol 3-kinase-mediated
pathway but requires neither S6K1 nor rpS6
phosphorylation. Mol. Cell. Biol. 22, 8101–8113 (2002).
75. Naora, H., Takai, I., Adachi, M. & Naora, H. Altered cellular
responses by varying expression of a ribosomal protein
gene: sequential coordination of enhancement and
suppression of ribosomal protein S3a gene expression
induces apoptosis. J. Cell. Biol. 141, 741–753 (1998).
76. Kim, J. et al. Implication of mammalian ribosomal protein S3
in the processing of DNA damage. J. Biol. Chem. 270,
13620–13629 (1995).
77. Boxer, L. M. & Dang, C. V. Translocations involving c-myc
and c-myc function. Oncogene 20, 5595–5610 (2001).
78. Coller, H. A. et al. Expression analysis with oligonucleotide
microarrays reveals that MYC regulates genes involved in
growth, cell cycle, signaling, and adhesion. Proc. Natl Acad.
Sci. USA 97, 3260–3265 (2000).
79. Boon, K. et al. N-myc enhances the expression of a large
set of genes functioning in ribosome biogenesis and protein
synthesis. EMBO J. 20, 1383–1393 (2001).
SAGE analysis of MYCN target genes in human
neuroblastoma cells reveals that most MYC target
genes are involved in ribosome biogenesis and
protein synthesis.
80. Menssen, A. & Hermeking, H. Characterization of the cMYC-regulated transcriptome by SAGE: identification and
analysis of c-MYC target genes. Proc. Natl Acad. Sci. USA
99, 6274–6279 (2002).
81. Iritani, B. M. & Eisenman, R. N. c-Myc enhances protein
synthesis and cell size during B lymphocyte development.
Proc. Natl Acad. Sci. USA 96, 13180–13185 (1999).
The direct overexpression of c-Myc during B-cell
development results in increased cell growth that is
independent of the cell cycle and correlates with an
increase in total protein synthesis in the cell.
82. Kim, S., Li, Q., Dang, C. V. & Lee, L. A. Induction of
ribosomal genes and hepatocyte hypertrophy by
adenovirus-mediated expression of c-Myc in vivo. Proc. Natl
Acad. Sci. USA 97, 11198–11202 (2000).
83. Iritani, B. M. Modulation of T-lymphocyte development,
growth and cell size by the Myc antagonist and
transcriptional repressor Mad1. EMBO J. 21, 4820–4830
(2002).
84. Piedra, M. E., Delgado, M. D., Ros, M. A. & Leon, J. c-Myc
overexpression increases cell size and impairs cartilage
85.
86.
87.
88.
89.
90.
91.
92.
93.
94.
95.
96.
97.
98.
99.
100.
101.
102.
103.
104.
105.
106.
107.
108.
109.
110.
differentiation during chick limb development. Cell Growth
Differ. 13, 185–193 (2002).
Schmidt, E. V. The role of c-myc in cellular growth control.
Oncogene 18, 2988–2996 (1999).
Kozma, S. C. & Thomas, G. Regulation of cell size in growth,
development and human disease: PI3K, PKB and S6K.
Bioessays 24, 65–71 (2002).
Di Cristofano, A., Pesce, B., Cordon-Cardo, C. & Pandolfi, P. P.
Pten is essential for embryonic development and tumour
suppression. Nature Genet. 19, 348–355 (1998).
Di Cristofano, A., De Acetis, M., Koff, A., Cordon-Cardo, C.
& Pandolfi, P. P. Pten and p27KIP1 cooperate in prostate
cancer tumor suppression in the mouse. Nature Genet. 27,
222–224 (2001).
Vogt, P. K. PI 3-kinase, mTOR, protein synthesis and cancer.
Trends Mol. Med. 7, 482–484 (2001).
Backman, S., Stambolic, V. & Mak, T. PTEN function in
mammalian cell size regulation. Curr. Opin. Neurobiol. 12,
516–522 (2002).
Maehama, T. & Dixon, J. E. The tumor suppressor,
PTEN/MMAC1, dephosphorylates the lipid second
messenger, phosphatidylinositol 3,4,5-trisphosphate.
J. Biol. Chem. 273, 13375–13378 (1998).
Myers, M. P. et al. The lipid phosphatase activity of PTEN is
critical for its tumor supressor function. Proc. Natl Acad. Sci.
USA. 95, 13513–13518 (1998).
Brown, E. J. et al. Control of p70 s6 kinase by kinase activity
of FRAP in vivo. Nature 377, 441–446 (1995).
Burnett, P. E., Barrow, R. K., Cohen, N. A., Snyder, S. H. &
Sabatini, D. M. RAFT1 phosphorylation of the translational
regulators p70 S6 kinase and 4E-BP1. Proc. Natl Acad. Sci.
USA 95, 1432–1437 (1998).
Isotani, S. et al. Immunopurified mammalian target of
rapamycin phosphorylates and activates p70 S6 kinase α
in vitro. J. Biol. Chem. 274, 34493–34498 (1999).
Sabatini, D. M. Interaction of RAFT1 with gephyrin required for
rapamycin-sensitive signaling. Science 284, 1161–1164
(1999).
Neshat, M. S. et al. Enhanced sensitivity of PTEN-deficient
tumors to inhibition of FRAP/mTOR. Proc. Natl Acad. Sci.
USA 98, 10314–10319 (2001).
Podsypanina, K. et al. An inhibitor of mTOR reduces
neoplasia and normalizes p70/S6 kinase activity in Pten+/−
mice. Proc. Natl Acad. Sci. USA 98, 10320–10325
(2001).
References 97 and 98 report that Pten-deficient
tumours show an increase in mTOR activation and an
upregulation in p70S6k activity. These tumours can be
effectively treated with the macrolide rapamycin, the
cellular target of which is mTOR.
Gingras, A. C. et al. 4E-BP1, a repressor of mRNA
translation, is phosphorylated and inactivated by the
Akt(PKB) signaling pathway. Genes Dev. 12, 502–513
(1998).
Sekulic, A. A direct linkage between the phosphoinositide
3-kinase-AKT signaling pathway and the mammalian target
of rapamycin in mitogen-stimulated and transformed cells.
Cancer Res. 60, 3504–3513 (2000).
Pullen, N. et al. Phosphorylation and activation of p70s6k by
PDK1. Science 279, 707–710 (1998).
Aoki, M., Blazek, E. & Vogt, P. K. A role of the kinase
mTOR in cellular transformation induced by the
oncoproteins P3k and Akt. Proc. Natl Acad. Sci. USA 98,
136–141 (2001).
Podsypanina, K. et al. Mutation of Pten/Mmac1 in mice
causes neoplasia in multiple organ systems. Proc. Natl
Acad. Sci. USA 96, 1563–1568 (1999).
Suzuki, A. High cancer susceptibility and embryonic lethality
associated with mutation of the PTEN tumor suppressor
gene in mice. Curr. Biol. 8, 1169–1178 (1998).
Gomez, M., Sampson, J. & Whittemore, V. The Tuberous
Sclerosis Complex (Oxford Univ. Press, 1999).
Kwiatkowski, D. J. A mouse model of TSC1 reveals sexdependent lethality from liver hemangiomas, and upregulation of p70S6 kinase activity in Tsc1 null cells. Hum.
Mol. Genet. 11, 525–534 (2002).
Inoki, K., Li, Y., Zhu, T., Wu, J. & Guan, K. L. TSC2 is
phosphorylated and inhibited by Akt and suppresses mTOR
signalling. Nature Cell Biol. 4, 648–657 (2002).
Gao, X. Tsc tumour suppressor proteins antagonize
amino-acid-TOR signalling. Nature Cell Biol. 4, 699–704
(2002).
Tee, A. R. Tuberous sclerosis complex-1 and -2 gene
products function together to inhibit mammalian target of
rapamycin (mTOR)-mediated downstream signaling. Proc.
Natl Acad. Sci. USA 99, 13571–13576 (2002).
Radimerski, T., Montagne, J., Hemmings-Mieszczak, M. &
Thomas, G. Lethality of Drosophila lacking TSC tumor
suppressor function rescued by reducing dS6K signaling.
Genes Dev. 16, 2627–2632 (2002).
VOLUME 3 | MARCH 2003 | 1 9 1
REVIEWS
111. Goncharova, E. A. et al. Tuberin regulates p70 S6 kinase
activation and ribosomal protein S6 phosphorylation. A role
for the TSC2 tumor suppressor gene in pulmonary
lymphangioleiomyomatosis (LAM). J. Biol. Chem. 277,
30958–30967 (2002).
112. Jaeschke, A. Tuberous sclerosis complex tumor
suppressor-mediated S6 kinase inhibition by
phosphatidylinositide-3-OH kinase is mTOR independent. J.
Cell Biol. 159, 217–224 (2002).
113. Lazaris-Karatzas, A., Montine, K. S. & Sonenberg, N.
Malignant transformation by a eukaryotic initiation factor
subunit that binds to mRNA 5′ cap. Nature 345, 544–547
(1990).
The first demonstration that a translation initiation
factor shows oncogenic properties when
constitutively overexpressed.
114. De Benedetti, A. & Harris, A. L. eIF4E expression in tumors:
its possible role in progression of malignancies. Int. J.
Biochem. Cell Biol. 31, 59–72 (1999).
115. Gingras, A. C., Raught, B. & Sonenberg, N. eIF4 initiation
factors: effectors of mRNA recruitment to ribosomes and
regulators of translation. Annu. Rev. Biochem. 68, 913–963
(1999).
116. Ni, J., Tien, A. L. & Fournier, M. J. Small nucleolar RNAs
direct site-specific synthesis of pseudouridine in ribosomal
RNA. Cell 89, 565–573 (1997).
117. Lafontaine, D. L., Bousquet-Antonelli, C., Henry, Y.,
Caizergues-Ferrer, M. & Tollervey, D. The box H + ACA
snoRNAs carry Cbf5p, the putative rRNA pseudouridine
synthase. Genes Dev. 12, 527–537 (1998).
118. Dokal, I. Dyskeratosis congenita in all its forms. Br. J.
Haematol. 110, 768–779 (2000).
119. Giordano, E., Peluso, I., Senger, S. & Furia, M. minifly, a
Drosophila gene required for ribosome biogenesis, J. Cell
Biol. 144, 1123–1133 (1999).
120. Zebarjadian, Y., King, T., Fournier, M. J., Clarke, L. &
Carbon, J. Point mutations in yeast CBF5 can abolish in vivo
pseudouridylation of rRNA. Mol. Cell. Biol. 19, 7461–7472
(1999).
121. Mitchell, J. R., Wood, E. & Collins, K. A telomerase
component is defective in the human disease dyskeratosis
congenita. Nature 402, 551–555 (1999).
192
| MARCH 2003 | VOLUME 3
122. Rudolph, K. L. et al. Longevity, stress response, and cancer
in aging telomerase-deficient mice. Cell 96, 701–712
(1999).
123. Decatur, W. A. & Fournier, M. J. rRNA modifications and
ribosome function. Trends Biochem. Sci. 27, 344–351
(2002).
124. Kullmann, M., Gopfert, U., Siewe, B. & Hengst, L. ELAV/Hu
proteins inhibit p27 translation via an IRES element in the
p27 5’UTR. Genes Dev. 16, 3087–3099 (2002).
125. Cornelis, S. et al. Identification and characterization of a
novel cell cycle-regulated internal ribosome entry site. Mol.
Cell 5, 597–605 (2000).
126. Hoang, C. & Ferre-D’Amare, A. R. Cocrystal structure of
a tRNA Psi55 pseudouridine synthase: nucleotide
flipping by an RNA-modifying enzyme. Cell 107,
929–939 (2001).
127. Vagner, S., Galy, B. & Pyronnet, S. Irresistible IRES.
Attracting the translation machinery to internal ribosome
entry sites. EMBO Rep. 2, 893–898 (2001).
128. Da Costa, L., Willig, T. N., Fixler, J., Mohandas, N. &
Tchernia, G. Diamond-Blackfan anemia. Curr. Opin. Pediatr.
13, 10–15 (2001).
129. Willis, A. E. Translational control of growth factor and protooncogene expression. Int. J. Biochem. Cell. Biol. 31, 73–86
(1999).
130. Spence, J. et al. Cell cycle-regulated modification of the
ribosome by a variant multiubiquitin chain. Cell 102, 67–76
(2000).
131. Strezoska, Z., Pestov, D. G. & Lau, L. F. Bop1 is a mouse
WD40 repeat nucleolar protein involved in 28S and 5.8S
rRNA processing and 60S ribosome biogenesis. Mol. Cell.
Biol. 20, 5516–5528 (2000).
132. Pestov, D. G., Strezoska, Z. & Lau, L. F. Evidence of p53dependent cross-talk between ribosome biogenesis and the
cell cycle: effects of nucleolar protein Bop1 on G(1)/S
transition. Mol. Cell. Biol. 21, 4246–4255 (2001).
A defect in ribosome rRNA processing can directly
affect cell-cycle progression through a p53dependent process. This is a hallmark study
proposing that a nucleolar stress affects cellular
proliferation through crosstalk between the ribosome
and the cell cycle.
133. Hidalgo, M. & Rowinsky, E. K. The rapamycin-sensitive
signal transduction pathway as a target for cancer therapy.
Oncogene 19, 6680–6686 (2000).
134. Decatur, W. A. & Fournier, M. J. RNA-guided nucleotide
modification of ribosomal and other RNAs. J. Biol. Chem.
278, 695–698 (2003).
135. Manning, B. D., Tee, A. R., Logsdon, M. N., Blenis, J. &
Cantley, L. C. Identification of the tuberous sclerosis
complex-2 tumor suppressor gene product tuberin as a
target of the phosphoinositide 3-kinase/akt pathway.
Mol. Cell 10, 151–162 (2002).
136. Potter, C. J., Pedraza, L. G. & Xu, T. Akt regulates growth by
directly phosphorylating Tsc2. Nature Cell Biol. 4, 658–665 (2002).
References 135 and 136 show that AKT
phosphorylates TSC2 and thereby inhibits its function
as negative regulator of p70S6k.
Acknowledgments
We are indebted to M. Barna for input, discussion and critical
reading of the review. We apologize to the many scientists
whose work we did not cite due to space constraints. This work
is supported by the National Cancer Institute, the Mouse Model
of Human Cancer Consortium (MMHCC) and the Leukemia
Lymphoma Society (LLS).
Online links
DATABASES
The following terms in this article are linked online to:
CancerNet: http://www.cancer.gov.search/
leukaemia | small-cell lung carcinoma
FlyBase: http://flybase.bio.indiana.edu/
brat
LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink
CDK2 | CDK4 | cyclin D1 | DKC1 | EEF1A1 | EEF2 | EGF | MYC |
NPM | nucleolin | p53 | PDK1 | PTEN | RB | TIF-IA | TIF-IB | TSC1 |
TSC2
OMIM: http://www.ncbi.nlm.nih.gov/Omim
Diamond–Blackfan anaemia | dyskeratosis congenita |
lymphangioleiomyomatosis | tuberous sclerosis
Access to this interactive links box is free online.
www.nature.com/reviews/cancer