Understanding p53: new insights into tumor suppression

Abstract

p53 (aka TP53) is a powerful tumor suppressor, and oncogenic transformation is induced when the ability of p53 to suppress tumorigenesis is compromised. p53 not only prevents tumorigenesis, but also tumor progression, that is, local invasion and distant metastasis. Recently, we showed that cytoplasmic p53 prevents RAS-driven invasion via alteration of actin cytoskeleton remodeling. This follows modulation of mitochondrial integrity. The transcriptional activity of p53 has been restored using small molecules; however, their success as cancer therapies is largely dependent on the status of downstream targets of p53. It is therefore important to elucidate the role of these downstream targets in p53 regulated tumor progression. With the recently described mechanism of tumor suppression highlighting a role of p53’s downstream targets in the regulation of actin cytoskeleton dynamics and lamellipodia formation, we suggest that potential therapeutic targets may be revealed within this mechanism that can be exploited in anticancer therapy.

Keywords::

• actin
• apoptosis
• invasion
• p53
• RAS

p53 suppresses tumorigenesis

p53 is a transcription factor that is well known for its wide range of functions, including the induction of apoptosis, senescence and cell cycle arrest [1]. In cells growing under normal conditions, p53 is maintained at low levels via a continual proteasomal degradation mediated by the E3 ligase, mouse double minute 2 homolog (MDM2). However, in response to stresses such as genotoxic damage, p53 is stabilized and activated, allowing it to induce the expression of its target genes to prevent tumorigenesis.

As a tumor suppressor, p53 also provides additional intrinsic mechanisms to prevent tumorigenesis. For example, it restricts the metabolic shift that commonly occurs in cancer cells, from oxidative phosphorylation to aerobic glycolysis. This occurs via an induction of the target genes TIGER and SCO2 [2]. p53 also attenuates glycolysis by downregulating the expression of glucose transporters [2,3]. This maintains energy provision from oxidative phosphorylation.

Targeting p53 in anticancer therapy

With crucial roles in tumor suppression, it is no surprise that a loss of p53 activity is associated with tumorigenesis. The role of p53 in tumor suppression is of great interest in the development of new anticancer therapies and indeed the reactivation of p53 by small molecule drugs has been clinically trialed. MDM2 inhibition is of particular interest, and there are a number of MDM2 antagonists currently in Phase I clinical trial for the treatment of various cancers [4]. Despite these agents needing to overcome intrinsic mechanisms that confer protection against apoptosis to cancer cells, these trials are yielding promising results. Synergistic effects have been noted when Nutlin-3, an MDM2 inhibitor that is known to arrest the cell cycle in a p53-dependent manner, was combined with established anticancer agents such as doxorubicin, vincristine or cisplatin, among others [5–7]. The most intriguing results in the development of such therapies have recently been described in an extensive review by Hoe et al. [4].

Despite early expectations that the MDM2 antagonists would function with limited genotoxicity, studies have since shown an abundance of cases of resistance acquired through the introduction of mutations within p53 [8,9]. TP53 is mutated in approximately 50% of human cancers. As the ultimate effect of reactivating dysfunctional p53 is the induction of apoptosis, it has been proposed that the MDM2 inhibitors may be vulnerable to similar resistance mechanisms as pro-apoptotic agents. Indeed, this has been observed in in vitro studies using the most common MDM2 inhibitor, Nutlin-3 [10,11]. Restoring wild-type activity to mutant p53, which otherwise prevents the proper folding, or disrupts its DNA-binding activity, by small molecules such as PRIMA-1MET, will therefore be effective [4].

Recent insights into p53 -mediated tumor suppression

Although reactivation of p53 is desired for the subsequent induction of apoptosis, p53 maintains multiple roles in the cell and it is not inconceivable that other roles could be exploited as a therapeutic target. Recently, our group revealed that cytoplasmic p53 prevents cell invasion via an alteration of actin cytoskeleton remodeling [12].

Oncogenic RAS is observed in approximately 30% of human cancers, and this produces an even poorer prognosis. A specific role of p53 is the regulation of RhoA activity and subsequent suppression of RAS-driven invasion [13,14], which is promoted via the induction of actin reorganization [15]. With cell invasion or migration driving cancer cell metastasis, this role of p53 holds great potential as a new target for anticancer therapy.

Cell migration is a particularly complex process, resulting from several interdependent processes that include the formation of integrin-based focal adhesion complexes and the reorganization of the actin cytoskeleton network [16]. Alterations to focal adhesion dynamics, as well as actin filament assembly, are known to promote cell invasion and subsequently enhance cancer progression [17].

We found that p53 suppresses RAS-mediated tumor progression by modulating distinct signaling processes that ultimately lead to the cleavage of β-actin, a reduction in filamentous actin and a reduction in the phosphorylation of p130 Crk-associated substrate at focal adhesions. This then impedes lamellipodia development, and hence, limits cell invasion.

Rather than acting directly on the cytoskeleton, p53 mediates the activation of high temperature requirement A2 (HtrA2; also known as Omi), a mitochondrial intermembrane space protein. This results from the accumulation of p53 in the cytoplasm following RAS transformation. Translocation of p38 MAPK into mitochondria is subsequently enhanced, without inducing apoptosis. p38 MAPK is required for phosphorylation of HtrA2/Omi and subsequently its protease activity [18].

Concurrently, oncogenic RAS promotes mitochondrial fragmentation, which causes HtrA2/Omi, to be released into the cytosol. This occurs as a result of mitochondrial outer membrane permeability (MOMP), which is commonly observed following the onset of apoptosis and the activation of Puma (aka BBC3) and Bax (aka BAX), two well-known products of p53 target genes [19]. In this case, however, MOMP was induced independently of apoptosis as well as induction of BBC3 and BAX. It was also induced in the absence of p53, with RAS-transformation alone promoting mitochondrial fragmentation. This was confirmed by the induction of mitochondrial fusion, which impeded the release of HtrA2/Omi.

Once inside the cytosol, HtrA2/Omi cleaves β-actin, leading to filamentous actin disassembly and a perturbation of lamellipodia formation. As modulation of the cytoskeleton occurred without the onset of apoptosis, this additional role of p53 may prove valuable, especially where cancer cells have retained or gained mechanisms that protect them from apoptosis.

Downstream targets of p53, which regulate actin cytoskeleton remodeling, may serve as potential targets for anticancer therapies

Cancer is a disease that has a vast range of causes and outcomes. Often a patient’s prognosis is dependent on the presence or absence of specific mutations in causative genes, or in proteins serving as small molecule targets. Although a large amount of research is underway into the development of small molecules that can reactivate dysfunctional p53, their success may be limited by the genetic state of downstream targets of p53. Indeed, a common feature in cancer is the modulation to apoptotic pathways, with the levels of key proteins such as Bax or Puma often modulated [20].

Here, we have reiterated the importance of identifying additional roles of p53 to determine new therapeutic targets that may work synergistically with current p53 targeted therapies. HtrA2/Omi, for example, may be a promising therapeutic target. Considering that the loss of HtrA2/Omi causes mitochondrial dysfunction, which is commonly observed in cancer cells, therapeutically increasing the activity of HtrA2/Omi may inhibit invasion of cancer cells without damaging normal cells. Drugs, which induce mitochondrial p38 MAPK translocation, may synergistically prevent cancer cell invasion, by not only activating HtrA2/Omi, but also attenuating cytoplasmic p38 MAPK function, which otherwise contributes to metastasis. Of course, such possibilities are only provided here as theoretical examples, and future research is needed to determine the viability of targeting and modulating these downstream targets of p53 in future drug discovery efforts.

Conclusion

Our recent findings correlate p53 activity with actin cytoskeleton dynamics and the suppression of RAS-driven invasion. Through the activation of HtrA2/Omi, p53 suppresses tumor progression in an apoptosis-independent mechanism. Among the downstream targets of HtrA2/Omi are components of the actin cytoskeleton and focal adhesions. Given the importance of cell invasion to cancer progression, and the crucial roles of the cytoskeleton in cell invasion, we propose that this role of p53 may serve as a potential target for future anticancer therapies.

We have, so far, shown the suppression mechanism of p53 for RAS-driven progression using cell-based and ex vivo experiments. Hence, the challenge is to further characterize the influence of p53 on the cytoskeleton in vivo and establish the genetic status of potential downstream targets of p53 in various cancer types. Although the actin cytoskeleton is crucial to nearly all cells, and cell migration is essential during processes such as wound healing or embryogenesis, it remains possible that subtle differences exist within cancer cells that can be exploited for the identification of therapy targets.

Financial & competing interests disclosure

This work was supported by the National Research Foundation, Singapore and the Ministry of Education, Singapore through the Mechanobiology Institute in the National University of Singapore. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.