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The Roles of P53R2 in Cancer Progression based on the new function ofmutant p53 and cytoplasmic p21

Bahman Yousefi, Mohammad Rahmati, Yasin Ahmadi

PII: S0024-3205(14)00165-9DOI: doi: 10.1016/j.lfs.2014.01.063Reference: LFS 13889

To appear in: Life Sciences

Received date: 18 December 2013Accepted date: 15 January 2014

Please cite this article as: Yousefi Bahman, Rahmati Mohammad, Ahmadi Yasin, TheRoles of P53R2 in Cancer Progression based on the new function of mutant p53 andcytoplasmic p21, Life Sciences (2014), doi: 10.1016/j.lfs.2014.01.063

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The Roles of P53R2 in Cancer Progression based on the new function of

mutant p53 and cytoplasmic p21

Authors’ names: Bahman Yousefi1, 2

, Mohammad Rahmati3, Yasin Ahmadi*

4

1 Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

2 Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran

3, 4 Department of Biochemistry and Clinical Laboratories, Faculty of Medicine, Tabriz

University of Medical Science, Iran

* Corresponding author: Yasin Ahmadi, Department of Biochemistry and Clinical Laboratories,

Faculty of Medicine, Tabriz University of Medical Science, Iran.

E-mail address: ahmadi.bchemistry@yahoo.com , phone: +98 9368647695, Fax: +98

8326221611

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Abstract

Although deregulated expression of p53R2, a p53 inducible protein and homologue of the R2

subunit of ribonucleotide reductase, has been detected in several human cancers, p53R2 roles in

cancer progression and malignancy still remains controversial. In this article, we present a

viable hypothesis about the roles of p53R2 in cancer progression and therapy resistance based

on the roles of cytoplasmic p21 and mutant p53. Since p53R2 can upregulate p21 and p21 in

turn has a dual role in cell cycle, hence p53R2 can play a dual role in cell cycle progression. In

addition, because p53 is the main regulator of p53R2, the mutant p53 may induce the expression

of p53R2 in some cancer cells based on the “keep of function” phenomenon. Therefore,

depending on the locations of p21 and the new abilities of mutant p53, p53R2 has dual role in

cell cycle progression. Since the DNA damaging therapies induce p53R2 expression through

induction of p53, p53R2 can be the main therapy resistance mediator in cancers with

cytoplasmic p21.

Key words: cancer progression; cell cycle; keep of function phenomenon; therapy resistance

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Introduction

P53r2 and ribonucleotide reductase

Human ribonucleotide reductase a rate limiting enzyme complex in synthesis of dNTP, is a

tetramer composed of two dissimilar homodimers including hRRM1 (R1) and hRRM2 (R2)

(Fontecave, Lepoivre, Elleingand, Gerez, & Guittet, 1998) . R1 and R2 are expressed

exclusively during the S-phase. Because of the long half-life of R1, its level is constant

throughout cell cycle and always in excess of the R2 level (Wang, Zhenchuk, Wiman, &

Albertioni, 2009; Zhang et al., 2011) . In G1-phase, R2 is degraded by cadherin 1/anaphase

promoting complex (Cdh1/APC) that binds to KEN box of R2 (Pontarin et al., 2007). P53R2 is

a homologue of R2 and its gene contains a p53-binding site in intron 1 and encodes a 351-

amino acid peptide that shows remarkable resemblance to R2 subunit of RR (Nakamura, 2004).

Since p53R2 does not have a KEN box, it is not degraded in G1-phase. Therefore, during G1,

p53R2 associates with R1 instead of R2 and subsequently provides dNTP for DNA repair in G1

(Chang et al., 2008; Pontarin, et al., 2007; Zhang, et al., 2011). While p53R2 expression is

regulated in a p53-dependent manner in result of DNA damage in G1 phase(Tanaka et al.,

2000), RRM2 expression is dictated by cell cycle-associated factors, such as nuclear factor Y

and E2f during S-phase(Chabes, Björklund, & Thelander, 2004). Thus, there are two

independent pathways which supply deoxyribonucleotides: i) through R2 in S-phase and ii)

through p53R2 for DNA repair in cells arrested in G1 or G2-phase(Yamaguchi et al., 2001).

The maximal level of p53R2 has been detected at G1/S transition (Wang, et al., 2009).

Furthermore, p53R2 upregulates P21 and downregulates cyclin D (Zhang, et al., 2011), causing

cell cycle arrest in G1 and providing both time and dNTP for the repair of damaged DNA. But

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in spite of these facts, deregulated expression of p53R2 has been found in various human cancer

cells. The relationship between p53R2 expression and a number of cancer cells has been listed

in table 1.

Table 1. The effect of p53R2 expression on cancer cell progression or therapeutic resistance.

Type of cancer cell p53R2 level

Remarks Reference

Colon adenocarcinoma High

p53R2 is negatively related to metastasis of

colon adenocarcinoma and high level of

p53R2 is correlated with markedly better

survival in CRC patients

(X. Liu et al., 2011; X. Liu

et al., 2007)

Squamous cell carcinoma High

High level of p53R2 is related to tumor

development and resistance against

chemo-radiation therapy

(Okumura et al., 2006)

Gastric cancer Basal (or

decreased)

There is not an association between stage,

grade and progression of gastric cancer cell

and p53R2

(Byun, Chae, Ryu, Lee, &

Chi, 2002)

Human oral cell carcinoma

cell lines, SAS (p53 wild-

type), HSC-4 (p53 mutant),

Ca9-22 (p53 mutant) and

human breast carcinoma

cell line, MCF7 (p53 wild-

type)

High

High expression of p53R2 was

significantly associated with tumor size,

lymph node metastasis and histological

differentiation and tumor was more

resistant to 5-FU

(Souichi Yanamoto et al.,

2005)

Oral squamous cell

carcinomas High

p53R2 was significantly associated with

tumor size, lymph node metastasis and

histological differentiation

(S. Yanamoto, Kawasaki,

Yoshitomi, & Mizuno,

2003)

Melanoma cancer High

p53R2 was significantly correlated with

the depth of invasion ,the tumor stage

and chemoresistance

(Matsushita et al., 2012)

Prostate cancer High p53R2 is associated with drug resistance (Devlin et al., 2008)

Non-small cell lung cancer High High level of p53R2 may be a marker of

the malignant potential of lung cancer

(URAMOTO, SUGIO,

OYAMA, HANAGIRI, &

YASUMOTO, 2006)

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Mutant p53, gain and “keep of function” phenomenon

Since mutant p53 (mt-p53) has been found in approximately 50% of human cancers, p53 was

named as guardian of genome (Levine, 2011). Wild-type (wt) p53 acts as a homotetramer

transcription factor that activates transcription of several hundred genes and regulates many

vital biological processes, including cell differentiation, proliferation, and apoptosis (Wei,

Zaika, & Zaika, 2011). While mutations in most other tumor suppressor genes results in loss or

aberrant synthesis of the gene product, most p53 mutations occur in DNA binding domain

(residues 100–300) (Soussi & Lozano, 2005), and therefore mutant p53 can gain new functions

in cell migration, invasion or metastasis (Muller, Vousden, & Norman, 2011). The

accumulation of mutant p53 in nucleus can exert a dominant negative role by oligomerization

with wt p53 expressed by the wt-allele (D.Michalovitz, 1991). Three scenarios have been

proposed to explain the effects of mutant p53 in tumor biology, which are not mutually

exclusive: i) mutations in p53 result in loss of tumor-suppressive functions of wt p53 solely; ii),

mutations of p53 may lead to loss of certain tumor-suppressive functions of wt p53, while

retaining and/or exaggerating other normal wt p53 function and iii), mt-p53 proteins can gain

truly neomorphic functions that promote tumor growth. Such activities of mt-p53 are commonly

attributed to one of two primary mechanisms: i) an interaction between mt-p53 and cellular

proteins, for instance through the inhibition of p63 and p73, which are responsible for the

induction of apoptosis (discussed below) or ii) mt-p53-mediated regulation of novel target

genes, e.g. mt-p53 proteins can up-regulate genes which inhibit apoptosis or promote

chemoresistance (Freed-Pastor & Prives, 2012) or can activate multidrug resistance genes

(ABCB1, ABCC1, ABCG1, and MVP), growth factor receptor genes (EGFR, bFGF, and

VEGF), and oncogenes (с-Myc, с-Fos, and Ras) (Denisov et al.).

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P21

One of the best known p53 targets genes is p21, a small 165 amino acid protein (also known as

WAF1, CIPl, SDIl and MDA-6) which regulates various cell cycle progression associated genes

such as cyclin E and cyclin A/CDK complexes to cause p53-dependent G1 growth arrest (Abbas

& Dutta, 2009; Gartel, Serfas, & Tyner, 1996). P21 mediates its functions through several

mechanisms: i) P21 binds to and inhibits CDK2 and CDK1 expression, arresting cell growth by

inhibition of phosphorylation of Rb by CDKs (Polager & Ginsberg, 2009) ; 2) P21 competes for

binding to proliferating cell nuclear antigen (PCNA) with DNA polymerase-δ and several other

proteins involved in DNA synthesis, thus directly inhibiting DNA synthesis (Cayrol, Knibiehler,

& Ducommun, 1998) and iii) p21 activates p21-activated kinases (Kumar, Gururaj, & Barnes,

2006). P21 disrupts the interaction between CDK and its substrates such as members of Rb

family (p107, p130, Rb) and CDC25 (a tyrosine phosphatase that dephosphorylates the cyclin

B-bound CDK1 that is essential for entry into mitosis) and leads to cell cycle arrest (Harbour &

Dean, 2000; Hartwell & Kastan, 1994) . Contrary to growth-inhibitory functions, recent

evidence show that cytoplasmic p21 has an important role in cell cycle progression and cell

survival (Perez-Tenorio et al., 2006), and protecting cell against of apoptosis (Asada et al.,

1999). Cytoplasmic p21 have been detected in many human malignancies and correlates

positively with aggressive tumors and poor prognosis, because it may acquire an anti-apoptotic

gain-of-function in the cytoplasm (Abbas & Dutta, 2009; Blagosklonny, 2002). The mechanism

of cytoplasmic localization of p21 remains to be studied, but it has been revealed that the

phosphorylation of P21 in Thr145 and Ser146 residues or truncation of nuclear localization

signal (NLS) of p21 can result in cytoplasmic localization of P21 (Goh, Coffill, & Lane, 2011;

Perez-Tenorio, et al., 2006). The main enzyme thought to be responsible for P21

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phosphorylation at these residues is Akt1, also known as PKB. Activation of PKB/Akt pathway

by the erbB2 receptor is partially responsible for phosphorylation of P21at these residues

(Abukhdeir & Park, 2008; Perez-Tenorio, et al., 2006). Phosphorylation of P21 by Akt/PKB at

Thr 145 in the PCNA-binding site disrupts its binding to PCNA (induces the cytoplasmic

accumulation of p21), whereas phosphorylation of Ser146 significantly increases the stability of

p21 protein (Cayrol, et al., 1998; Li, Dowbenko, & Lasky, 2002) . Akt can also be activated

through other genetic alterations, including phosphoinositide 3-kinase activation from

oncogenic mutations of PIK3CA, PTEN loss or HER2/neu (ERBB2) amplification (Abukhdeir

& Park, 2008). Cytoplasmic p21 inhibits a number of proteins involved in apoptosis such as

procaspase 3 (phosphorylated p21 by PKB binds to procaspase 3 and procaspase3/p21 complex,

induces resistance to Fas-mediated apoptosis) , caspase 8, caspase 10, stress-activated protein

kinases (SAPKs) and apoptosis signal-regulating kinase 1 (Abbas & Dutta, 2009; Schepers,

Geugien, Eggen, & Vellenga, 2003). Furthermore, P21 can upregulate the genes encoding anti-

apoptotic factors. P21 also downregulates the pro-apoptotic genes by MYC and E2F1 through

direct binding and inhibition of their transactivation functions (Abbas & Dutta, 2009).

The correlation of p53R2, p21 and mt p53 in cancer progression

As mentioned earlier, deregulated expression of p53R2 has been found in several human cancer

cells and its expression is associated with cancer cell progression or tumor suppressor roles.

P53R2 and other proteins involved in cell cycle progression and cell cycle arrest are under tight

control and they have precise and balanced function in the regulation of cell cycle. Cells employ

various strategies to survive and to control their fate. Upon encounter with a danger such as

DNA damage, cells launch specific processes such as cell cycle arrest and apoptosis.

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P53R2 is one of the most tangible demonstrations exhibiting cell tendency to survive. In early

G1 phase, cell exposure to stressors such as DNA damage drives the expression of p53R2.

By cell cycle progression, the p53R2 expression is increased and in G1/S transition it reaches

maximum level. P53R2 overexpression downregulates cyclin D and upregulates P21 and P21

activates CDK4, 6/cyclin D (Zhang, et al., 2011) and subsequently these CDKs activate E2f

family by phosphorylation of Rb. E2fs contributes to cell cycle progression and proliferation

(Wu & Levine, 1994) Also, p53R2 directly provides dNTPs for DNA repair. With cell cycle

progression, the positive effects of p53R2 in activation of CDK4, 6/ cyclin D (by P21)

overcomes its negative effects (downregulation of D cyclin) on these CDKs. Furthermore,

although p21 inactivates CDK2, high levels of CDK4, 6/ cyclin D in G1/S transition activates

CDK2 and therefore causes cell entry to S-phase (Zhang, et al., 2011). Finally phosphorylation

of P21 at Ser130 by CDK2/ cyclin E promotes its binding to SKP2, resulting in ubiquitylation

and subsequently proteolysis of P21, and thus promotes cell cycle progression at the G1/S

transition. In fact, at the beginning of G1 phase, cell slows down its progression speed in order

to find and eliminate the defects in DNA sequence. Therefore, p53R2 plays its role in the

progression of cell cycle in a stepwise and time dependent manner.

Cancerous cells exploit natural cellular mechanisms to supply their high demands for

continuous proliferation. In the case of p53R2, this protein plays dual role and it may be anti-

carcinogenic in wt cell or play a role in line with cancer cell cycle progression and

carcinogenesis. Cancer cells exploit the ability of p53R2 to provide dNTPs for repairing their

damaged DNA resulting from chemo or radiotherapy. likewise and more importantly, p53R2

upregulates P21, and cytoplasmic P21 in turn, acts as a carcinogen and anti-apoptotic agent;

thus, upregulation of cytoplasmic P21 by p53R2 increases cell cycle progression and cell

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proliferation and subsequently induces progression of cancer. Akt is one of the most important

factors involved in the cytoplasmic localization of P21 (Li, et al., 2002). In Akt-overexpressing

cancers, p53R2 may play a tumorigenic role and promote drug resistance against anti-cancer

drugs. In cancer with cytoplasmic P21, p53R2 plays a positive role in cancer progression,

chemoresistance, and poor prognosis. In addition, p53R2 binds to P21 before DNA damage.

The binding between p21 and p53R2 decreases in response to UV(Xue et al., 2007); thus, after

radiotherapy or other DNA damaging therapies, the level of free p21 and p53R2 increases in

nucleus (see Fig.1).

Figure 1. After DNA damage, p53 has been induced.p53 activates transcription of p21 and p53R2. P53R2 more activates

transcription of p21.in absent of Akt, induced p21 binds to Cdks and activate Rb. Then Rb binds to E2fs and inactivates E2fs

that result in arresting cell cycle progression. Besides, p21 binds to PCNA and disrupt the linkage between PCNA and DNA

polymerase. Therefore inactivates DNA proliferation. Radiation separates p53R2 and p21 (right side). When Akt exists, it

phosphorylates and transmits p21 to cytoplasm that inhibits apoptosis.

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Since the main regulator of p53R2 is p53 (Wang, et al., 2009), in order to investigate roles and

levels of p53R2 in cancer cell progression, cancer cells should be categorized in two groups of

mt-p53 tumors and non mt-p53 tumors. Thus, mt-p53 may gain new functions or keep original

function (Freed-Pastor & Prives, 2012). It is possible that high level of mt-p53 can gain a higher

capacity for activation (gain of function) or at least mt-p53 can maintain the ability to activate

the expression of p53R2 (keep of function).

As well, until recently, the high levels of mt-p53 in some cancer cells were hypothesized to be

associated with the inability of mt-p53 to activate transcription of mdm2 (loss of function

phenomenon). In contrast to this hypothesis, in the mt-p53 knock-in mouse models (in one or

both p53 alleles),although all tissues contained the mutant allele, these mice did not accumulate

mt-p53 in most normal tissues, while levels of mt-p53 were frequently high in the tumors

(Freed-Pastor & Prives, 2012). Therefore it can be inferred that the overexpression of mt-p53

cannot be explained based on loss of function phenomenon.

The high levels of mt-p53 in cancer cells are explainable based on the roles of E2fs which

control the expression of three gene classes: i) genes involved in G1/S transition, such as the

cyclins E and A, the c-myc gene and the gene encoding pRb and the related p107; ii) genes

involved in synthesis and replication of DNA such as dihydrofolate reductase, Cdc6, and

thymidine kinase and iii) E2fs induce the expression of the genes encoding p19 ARF. E2fs

induce cell cycle progression and cell proliferation through upregulation of the first and second

classes of genes. Likewise, E2fs induce expression of ARF, and ARF increases the level of p53

(Garrett, 2001).. P53 inhibits cell cycle progression and proliferation. Since the levels of E2fs

positively corresponds to the rate of cell cycle progression and ARF level, therefore ARF act as

cell proliferation sensor.

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In cancer cells, the high rate of proliferation activates ARF transcription. In mt-p53 cancer cells,

the increased level of ARF dissociates mt-p53 from mdm2 and subsequently increases the levels

of mt-p53 in cancer cell. High levels of mt-p53 may overexpress p53R2 and cancer cells exploit

the positive effects of p53R2 on cell cycle progression to their own benefit. The roles that

p53R2 plays in wt p53 cancer cell is similar to those in mt-p53 cancer cells and p53R2 roles in

wt cell is dependent on p21 locations, although mutations in p53 complicates the roles of p53R2

in cancer progression by increasing p53R2 expression. Through gain of function phenomenon,

mt-p53 is capable of recruiting some genes product such as NY-F (K. Liu, Ling, & Lin, 2011) in

order to express their untraditional genes such as R2 small subunit of RR complex and thereby,

mt-p53 can provide dNTPs required for cancer cell progression. Therefore some mt-p53 may

provide dNTP in p53R2-independent pathway and the tumors with such character, bypass the

negative effects of p53R2 on cancer progression in providing dNTP pathway.

Conclusion

Before formation of cancer cell, p53R2 provides dNTPs for DNA repair and increases expression

of P21 while decreasing the expression of cyclin D in wt cell to arrest cell cycle in order to repair

damaged DNA. After formation of malignancy and their increasing demands for nutrients and

support, p53R2 may contributes to cancer cell progression especially when p21 presents in

cytoplasm.

Future work

With regard to the relationship between p53r2 and p21 to more investigate the roles of p53r2 in

cancer progression and (DNA damaging) therapy resistance, it is better to focus on the factors

that localize p21 in cytoplasm and among these, Akt can be the most important factor to

investigation.

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Conflict of interest statement

The authors declare that there are no conflicts of interest

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Graphical abstract