1 ribosome biogenesis and control of cell - cancer research

1

Ribosome biogenesis and control of cell proliferation: p53 is not alone.

Authors: Giulio Donati1,*, Lorenzo Montanaro1and Massimo Derenzini2§.

Running Title: p53-independent control of cell proliferation

Key words: ribosome biogenesis, cell proliferation, p53, cell cycle control, cancer

Affiliations: 1 Department of Experimental Pathology, Alma Mater Studiorum - Bologna

University, Bologna Italy. 2Clinical Department of Radiological and Histocytopathological

Sciences, Alma Mater Studiorum - Bologna University.

* Current address: Bellvitge Biomedical Research Institute (IDIBELL)-Hospital Duran i Reynals,

Barcelona, Spain

§ corresponding author: 2Clinical Department of Radiological and Histocytopathological Sciences,

Alma Mater Studiorum - Bologna University, Via Massarenti, 9 40138 Bologna Italy. E-mail:

[email protected]

Word Count: 2681

Figure number: 2

Research. on November 18, 2018. © 2012 American Association for Cancercancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on January 26, 2012; DOI: 10.1158/0008-5472.CAN-11-3992

http://cancerres.aacrjournals.org/

2

Abstract

Cell growth is a pre-requisite for cell proliferation, and ribosome biogenesis is a limiting factor for

cell growth. In mammalian cells, the tumour suppressor p53 has been shown to induce cell cycle

arrest in response to impaired ribosome biogenesis. Recently, p53-independent mechanisms of cell

cycle arrest in response to alterations of ribosome biogenesis have been described. These findings

provide a rational basis for the use of drugs that specifically impact ribosome biogenesis for the

treatment of cancers lacking active p53, and extend the scenario of mechanisms involved in the

relationship between cell growth and cell proliferation.




3

Introduction

Cell growth (increase in cell mass), and cell proliferation (increase in cell number), are two tightly

linked phenomena. In cells stimulated to proliferate, a progressive increase in cell constituents

occurs before division ensuring appropriately sized daughter cells (1). Cell growth is the

consequence of an enhanced stimulation of protein synthesis that characterizes proliferating cells.

The increased demand for protein synthesis is accomplished by changes in the rate of ribosome

biogenesis, and ribosome biogenesis is the major metabolic effort in a proliferating cell

(2). Ribosome biogenesis is the result of a series of coordinated steps occurring in the nucleolus.

These include the transcription of ribosomal genes by RNA polymerase I (Pol I) to produce the 47S

rRNA precursor, the modification and processing of this transcript to generate the mature 18S, 5.8S,

and 28S rRNA, the import to the nucleolus of 5S rRNA and ribosomal proteins (RPs), the assembly

of the rRNAs with the RPs to form the large 60S and the small 40S subunits of the mature

ribosome, and the export of ribosomal subunits to the cytoplasm (3). Transcription of ribosomal

genes requires the assembly of a specific multiprotein complex at the rDNA promoter containing

Pol I and, in mammals, at least three other basal factors: the Transcription Initiation Factor I (TIF-I)

A, the Selectivity Factor 1 (SL1), and the Upstream Binding Factor (UBF) (reviewed in 4) (See

Fig.1).

It is well established that at the end of G1-phase, the so-called restriction point defines a limit

beyond which the cell is committed to divide, independent of growth (5). Therefore, it is within the

G1 phase that a proliferating cell has to perform its effort of supplying the components necessary

for the transit from G1 to S phase, and it is at the end of G1 phase that the cell must determine

whether this effort has been successfully accomplished. Data indicate that it is the amount of

ribosomes produced that controls the G1-S phase transition thus regulating the cell cycle

progression. In partially hepatectomized mice with induced conditional deletion of 40S ribosomal

protein S6, hepatocytes with hindered ribosome production but not hindered protein synthesis failed

to enter the S phase (6). Drug-induced stimulation or inhibition of rRNA synthesis caused an

accelerated or delayed G1/S-phase progression, respectively, in rat hepatoma cells, due to an

accelerated or delayed achievement of the appropriate amount of ribosomes during the G1 phase

(7). Regarding the mechanism that regulates the cross-talk between ribosome biogenesis and cell

cycle progression there is a general consensus on the key role played by the tumour suppressor p53.

In fact, as described more extensively below, it has been clearly demonstrated that altered ribosome

biogenesis is responsible for p53 stabilization and, therefore, for the arrest of cell cycle progression




4

(reviewed in 8-10). On the other hand, very recent data indicate that p53-independent mechanisms

are also activated that hinder proliferation as a consequence of altered ribosome biogenesis (11-

15).

Ribosome biogenesis and the control of cell cycle progression: the p53 paradigm.

Defects in ribosome production cause p53 stabilization and induce a marked p53-dependent

inhibition of cell proliferation (10). It is worth recalling that in proliferating mammalian cells the

G1-S phase restriction point can be overwhelmed by the activation of the E2F transcription factor

family (16), which regulate the expression of genes whose products are necessary for the synthesis

of DNA, and therefore for the progression from G1 to S phase. The retinoblastoma protein (pRB),

the product of the tumour-suppressor RB1 gene, in its active hypophosphorylated form, is bound to

E2Fs and prevents them from inducing E2F target genes. In the hyperphosphorylated form, pRB no

longer binds to E2Fs, which can then activate their target genes. Phosphorylation of pRB is

triggered in the early G1 phase by cyclin D-Cdk-4 and -6 complexes and is completed at the end of

the G1 phase by cyclin E-Cdk-2 complexes. pRB phosphorylation is hindered by Cdk inhibitors:

Cdk-4 and Cdk-6 are inhibited mainly by p16Ink4a whereas Cdk-2 is negatively regulated by

p21(Cip1) and p27(Kip1) (Fig. 2a). p53 stabilization, occurring in response to a variety of cellular

stresses, leads to induction of the Cdk-2 inhibitor p21, inhibition of cyclin E/Cdk2 complexes, and

pRB-dependent cell cycle arrest at the G1/S phase check-point (16).

The mechanism of p53 stabilisation after perturbation of ribosome biogenesis is apparently the

consequence of changes in functional and physical interactions of the tumour suppressor with

MDM2. MDM2 negatively controls p53 activity in two ways: by binding to the protein and

interfering with its transactivation activity, and by facilitating p53 proteasomal degradation thereby

acting as an E3 ubiquitin ligase (17). As a consequence of altered ribosome biogenesis, several

ribosomal proteins no longer used for ribosome construction bind to MDM2 and relieve its

inhibitory activity toward p53. In fact, it was shown that the ribosomal proteins RPL5 (18), RPL11

(19-21), RPL23 (22,23) and RPS7 (24), bind MDM2, thus inducing p53 stabilisation by inhibiting

MDM2’s E3 ubiquitin ligase function (also reviewed in 10). The inhibition of rRNA transcription,

induced either by drugs such as actinomycin D, 5-fluorouracil, mycophenolic acid, or by depletion

of essential Pol I complex components (10, 15, 25), stabilizes p53 by causing RP-induced

inactivation of MDM2. These data are consistent with a mechanism by which altered ribosome

biogenesis inhibits cell proliferation in a strictly p53-dependent manner through the activation of

the ribosomal proteins-MDM2-p53 pathway.




5

p53-independent mechanisms

The regulation of cell cycle in response to defects in ribosome biogenesis, far from being exclusive

to mammalian cells, also occurs in organisms where p53 does not regulate the cell cycle, or is not

even present. In the metazoan Drosophila Melanogaster, p53 does not regulate p21 transcription,

nor the cell cycle (26) . In this organism, haploinsufficiency of several RP genes gives rise to the

Minute phenotype. Cells from Minute mutants are the same size as wild type, but their proliferation

rate is reduced (27), implying the existence of a mechanism controlling cell cycle progression in

response to hampered ribosome biogenesis. Depletion of the RNA polymerase I co-factor TIF-IA

induces a p53-independent proliferation arrest in Drosophila cells (28). Furthermore, in the yeast S.

cerevisiae, where no p53 homologue has been found, defective ribosome biogenesis delays the cell

cycle progression to S phase before any variation in protein synthesis capacity ensues (29).

The presence of a p53 independent pathway regulating the relationship between ribosome

biogenesis and cell proliferation in mammalian cells was first suggested by the observation that

selective inhibition of rRNA synthesis by actinomycin D was able to induce a perturbation of cell

cycle progression also in cells silenced for p53 expression, although milder than that occurring in

the presence of p53 (30). In the past two years some light has been shed on how altered ribosome

biogenesis can hinder cell proliferation in a p53-independent way. A good example is constituted by

pescadillo depletion. The pescadillo nucleolar protein plays a role in the processing of pre-rRNA

molecules during assembly of 60S ribosomal subunits, through formation of the PeBoW complex

with Bop1 and WDR12 proteins (11). Depletion of pescadillo inhibits ribosome biogenesis, and,

similar to other mechanisms that hinder ribosome production, stabilises p53 leading to cell cycle

arrest (11). However, pescadillo gene knockdown impaired cell proliferation in cells harbouring

mutated p53, indicating that in these cells, the observed cell cycle control mediated by pescadillo

protein was p53-independent (13). In fact, pescadillo depletion resulted in decreased expression of

cell cycle protein cyclin D1 and up-regulation of the CDK inhibitor p27, with the consequent

marked reduction of pRB phosphorylation (13).

Another p53-independent mechanism of cell proliferation arrest induced by impaired ribosome

biogenesis was demonstrated by the down-regulation of PIM1 expression caused by ribosomal

protein deficiency and various other ribosomal stressors (14). PIM1 is a constitutively active

serine/threonine kinase regulated by cytokines, growth factors and hormones that has been shown to

reduce the activity of p27Kip1 and increase its degradation (31). Furthermore, PIM1 kinase has

been shown to interact with the ribosomal protein RPS19 and it has been demonstrated that

depletion of RPS19 or other ribosomal proteins caused increased PIM1 kinase proteasome




6

degradation (14). The down-regulation of PIM1 kinase stabilized and activated p27Kip1, thus

causing a block in cell cycle progression regardless of p53 status (14).

The data reported above indicate that changes in the biogenesis of single ribosome subunit hinder

cell proliferation in cells with activated or inactivated p53. Moreover, data have recently been

reported showing that specific inhibition of rRNA transcription by depletion of POLR1A hindered

cell cycle progression in cancer cell lines with inactivated p53 (15). This was due to the fact that the

inhibition of rRNA synthesis decreased the expression of E2F-1. Normally, E2F-1 is protected from

proteasome-mediated degradation by the interaction with MDM2, which prevents the binding of

other E3 ligases responsible for E2F-1 ubiquitination (32). The inhibition of rRNA synthesis

releases the ribosomal protein L11, which, by binding to MDM2, prevents its stabilising function on

the E2F-1 protein (15). The down-regulation of the E2F-1 protein caused a reduction in the

expression of the E2Fs target genes that are necessary for the entry and progression through the S

phase. RB1 silencing rescued the expression of these genes completely and prevented the effect of

the rRNA synthesis inhibition on cell proliferation. The inhibition of rRNA synthesis is not always

associated with a down-regulation of E2F-1. In fact, actinomycin D, cisplatin and etoposide – drugs

that inhibit rRNA transcription – caused an accumulation of E2F-1 protein (33). However, these

drugs are also DNA-damaging agents, and it has been demonstrated that these agents increase E2F-

1 half-life and its transcriptional activity (33). Importantly, reducing the synthesis of rRNA by TIF-

IA silencing, i.e. a non DNA-damaging procedure to inhibit rRNA transcription, reduced the

expression of the E2F-1 protein (15).

Finally, another mechanism defining the relationship between ribosome biogenesis and cell

proliferation in cells with inactive p53 is the down-regulation of c-Myc in response to ribosome

stress. c-Myc, in addition to stimulating cell proliferation, controls all the steps of ribosome

biogenesis: it increases Pol I activity by facilitating the recruitment of SL1 to promoters, stimulates

ribosomal protein synthesis by increasing Pol II transcription, and enhances Pol III transcription by

activating TFIIIB (34). It was found that, in response to ribosome biogenesis inhibition, RPL11

binds to c-Myc reducing its transcriptional activity, and to c-Myc mRNA promoting its degradation

(12). The RPL11-mediated down-regulation of c-Myc reduced cell proliferation, and this effect was

also observed in p53-null cells, thus demonstrating another p53-independent mechanism of

proliferation control (12).

In conclusion, there is now evidence that ribosomal stress hinders cell proliferation in mammalian

cells with and without p53. The p53-dependent and –independent mechanisms by which ribosome

biogenesis controls cell cycle progression are schematically summarised in Fig. 2.




7

Ribosome biogenesis inhibition and chemotherapy of p53-deficient cancer

The disruption of p53 function occurs in more than 50% of human tumours, thus representing the

most frequent gene alteration in cancers (35). Commonly used chemotherapeutic agents in human

cancer induce some cell stress, which activates the p53-mediated cell cycle blockage and/or

apoptosis. Therefore, tumours without functioning p53 are less sensitive to cytostatic and cytotoxic

drug treatment than those harbouring wild type p53 (36). The p53-independent control of ribosome

biogenesis on cell proliferation may explain some recent data demonstrating an anti-proliferative

effect of Pol I complex inhibitors on cancer lacking functional p53. Indeed, a study conducted by

Drygin and colleagues (37) showed that targeting factors of the rDNA transcription complex

represents a promising strategy for chemotherapeutic treatment of cancers, both p53 proficient and

deficient. During a screening assay for agents that selectively inhibit Pol I transcription relative to

Pol II transcription, CX-5461 (Cylene Pharmaceuticals Inc.) a potent small-molecule that

selectively inhibits Pol I–driven transcription was identified (37). It was shown that CX-5461

disrupts the binding of the SL1 transcription factor to the rDNA promoter, thus preventing the

initiation of rRNA synthesis by the Pol I multiprotein complex. In fact, SL1 mediates specific

interactions between the rDNA promoter region and the Pol I enzyme complex by recruiting Pol I,

together with a series of Pol I–associated factors, to rDNA. CX-5461 exhibited anti-proliferative

activity in numerous cancer cells in vitro, and, importantly, this occurred in wild-type and mutant

p53 cell lines. In this study, the authors found that the mechanisms leading to cell death after

inhibition of rRNA synthesis involved autophagy and senescence. Unfortunately, potential

mechanisms affecting the control of cell cycle progression were not investigated. However,

accordingly to the results obtained by Pol I and TIF-IA depletion, it is likely that the inhibitory

effect on proliferation of cells lacking active p53 observed after CX-5461 treatment might be

mediated by a down-regulation of E2F-1.

Perspectives

Studies on p53-independent mechanisms regulating the relationship between cell growth and cell

cycle progression are expected to not only increase our knowledge of the homeostatic control of cell

proliferation, but also reveal new aspects of the process of tumourigenesis and potential

chemotherapeutic therapies for p53-negative cancers.

The stabilisation of p53 following ribosomal stress plays an important role against neoplastic

transformation (38). An altered ribosome biogenesis may be responsible for a failure in the p53

activation pathway, and this may facilitate tumour onset (39). Similarly, functional inhibition of

p53-independent mechanisms for opposing cell cycle progression after ribosomal stress could




8

down-regulate the tumour suppressor potential of the cell. Experimental studies of the effect of

alterations of factors involved in these mechanisms (such as Pim1, pescadillo, Bop1) on

tumourigenesis, together with the analysis of the integrity of their function in human tumours could

possibly point out their involvement in the process of neoplastic transformation and identify new

targets for therapeutic applications.

The results reported in this review strongly encourage studies to develop inhibitors of ribosome

biogenesis that down-regulate E2F-1 expression. Furthermore, considering the role of pRB in

controlling E2F-1 function, it might be of interest to combine the ribosome biogenesis inhibitors

with drugs that hinder the activity of the CDKs. The resulting reduction of E2F-1 expression,

together with the increased E2F-1 binding to hypophosphorylated pRB can be reasonably expected

to have a very strong impact on cell proliferation in cancers lacking active p53. A similar

chemotherapeutic approach should be highly effective on those cancers in which this transcription

factor is known to exert a major role in the biology of the transformed cells. For example, aberrant

E2F-1 activity is indeed a critical factor for the initiation and progression of B-cell Burkitt’s

lymphoma (40) and for the emergence of castration-resistant prostate cancer (41).

The use of drugs targeting the CDKs in combination with ribosome biogenesis inhibitors that down-

regulate E2F-1 expression may be also very useful for treatment of cancers characterised by Myc

overexpression, which is frequently observed in human tumours of different origin. In fact, these

drugs may exert a p53-independent anti-proliferative activity by both down-regulating c-Myc

mRNA expression as a consequence of an increased availability of RPL11 after the inhibition of

rRNA synthesis, and by reducing E2F-1 expression and activity.

Acknowledgements: We thank Dr. Angela Drew for critically editing the manuscript.

Grant Support: This work was funded by the Pallotti legacy for cancer research and RFO funds

from Bologna University to LM and MD. GD was supported by a grant fellowship by the Vanini

Cavagnino Legacy (Centro Interdipartimentale per le Ricerche sul Cancro “G.Prodi”, Bologna

University.




9

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1

Figure legends

Fig. 1. Schematic representation of the main processes involved in ribosome biogenesis. One

ribosomal gene unit is repeated hundreds of times in the genome and includes the promoter and the

transcribed region. The major basal factors binding to the promoter region and required for

ribosomal genes transcription are shown in the inset. After transcription, the 47S pre-rRNA is

processed. The mature rRNAs are assembled with ribosomal proteins (RPs) to generate mature

ribosomal sub-units. In particular 18S is assembled with small subunit RPs to generate the 40S

small ribosomal subunit, while 5.8S and 28S rRNA are assembled with 5S rRNA, (which is

transcribed by PolIII in the nucleus), and with large subunit RPs to form the 60S large ribosomal

subunit. Mature 40S and 60S subunits are then exported to the cytoplasm to constitute the

ribosomes.

Fig. 2. a) Schematic and simplified representation of the mechanisms leading to the cell cycle

progression from G1 to S phase. After cell cycle entry cyclin D-Cdk-4 and -6 and cyclin E-Cdk-2

complexes phosphorylate pRB during G1 phase. Phosphorylation of pRB leaves E2F1 free to

activate the E2F1 target genes whose products are necessary for entry and progression through S

phase.

b) p53-dependent cell cycle arrest after ribosomal stress. Regardless of the nature of ribosomal

stress, an increased availability of RPs inactivates MDM2 for p53 digestion. Accumulated p53

induces transcription of p21 that, in turn, hinders pRB phosphorylation by cyclin-dependent

kinases, thus blocking E2F-1 activity.

c) p53-independent cell cycle arrest after ribosomal stress. Alterations in ribosomal assembly and

ribosomal processing increase the level of the cyclin-dependent kinase inhibitor p27 thus reducing

pRB phosphorylation. This should lead to a decreased activation of E2F-1. Changes of ribosomal

biogenesis caused by inhibition of factors constituting the complex of rDNA gene promoter

increases RP availability. RP binding to MDM2 lowers its protective effect on E2F-1. The amount

of E2F-1 is therefore reduced by proteasome-mediated digestion with a consequent reduced

activation of the E2F-1 target genes.




Published OnlineFirst January 26, 2012.Cancer Res Giulio Donati, Lorenzo Montanaro and Massimo Derenzini alone.Ribosome biogenesis and control of cell proliferation: p53 is not

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1 ribosome biogenesis and control of cell - cancer research

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