The role of RAD9 in tumorigenesis

Abstract

RAD9 regulates multiple cellular processes that influence genomic integrity, and for at least some of its functions the protein acts as part of a heterotrimeric complex bound to HUS1 and RAD1 proteins. RAD9 participates in DNA repair, including base excision repair, homologous recombination repair and mismatch repair, multiple cell cycle phase checkpoints and apoptosis. In addition, functions including the transactivation of downstream target genes, immunoglobulin class switch recombination, as well as 3′–5′ exonuclease activity have been reported. Aberrant RAD9 expression has been linked to breast, lung, thyroid, skin and prostate tumorigenesis, and a cause–effect relationship has been demonstrated for the latter two. Interestingly, human RAD9 overproduction correlates with prostate cancer whereas deletion of Mrad9, the corresponding mouse gene, in keratinocytes leads to skin cancer. These results reveal that RAD9 protein can function as an oncogene or tumor suppressor, and aberrantly high or low levels can have deleterious health consequences. It is not clear which of the many functions of RAD9 is critical for carcinogenesis, but several alternatives are considered herein and implications for the development of novel cancer therapies based on these findings are examined.

Introduction

Cells bear a number of processes critical for maintaining genomic integrity when normal metabolic events or exposure to certain exogenous agents lead to DNA damage and the potential hindrance of DNA replication, transcription or other important cellular mechanisms. If damage is left unrepaired or is misrepaired, deleterious consequences, such as the development of cancer, can ensue (Kastan and Bartek, 2004). Cells routinely maintain the genome through pathways that directly repair damaged DNA or transiently delay cell cycle progression to prevent entry into critical phases such as S or M with damage and allow extra time for repair (Lazzaro et al., 2009; Fortini and Dogliotti, 2010). In contrast, apoptosis (i.e. programmed cell death) can be induced and result in the destruction of cells containing excessive irreparable damage (Bitomsky and Hofmann, 2009; Fortini and Dogliotti, 2010). Therefore, proteins that function in these processes are critical for the cell, and aberrant expression or activity could lead to cancer. RAD9 functions in multiple pathways that contribute to genome integrity, including DNA repair, cell cycle checkpoint control and apoptosis, and thus it is not surprising that aberrant RAD9 expression is linked to cancer. Nevertheless, the exact functions of RAD9 responsible for the development of cancer have not been defined. This article will examine the role of RAD9 in tumorigenesis, in the context of the known myriad activities of the protein.

The many activities and functional domains of RAD9

The RAD9 gene is evolutionarily conserved and orthologs have been isolated from a wide array of organisms, including yeast, fly, chicken, worm, mouse and human (Lieberman, 2006). One or both of the mammalian proteins were shown to demonstrate roles in maintaining genomic stability, DNA damage resistance, cell cycle checkpoint control, base excision repair, homologous recombination, mismatch repair, apoptosis, transactivation of downstream target genes, 3′–5′ exonuclease activity, regulation of ribonucleotide synthesis, co-repression of androgen-induced androgen receptor transactivity, immunoglobulin class switch recombination relative to antibody production and embryogenesis (Lieberman, 2006; Pandita et al., 2006; He at al., 2008; An et al., 2010; Greer Card et al., 2010). How this multitude of functions is regulated and coordinated remains unclear, although the phosphorylation status of RAD9, and differential interactions with a subset of known protein-binding partners likely exert considerable influence on the activity of the protein.

Figure 1 depicts the known functional domains and protein–protein interaction sites of human RAD9. Additional functions and protein interactors beyond what is illustrated in Figure 1 are known, but the relevant RAD9 amino acid residues have not been localized. RAD9 is thought to perform many of its activities as part of the RAD9–HUS1–RAD1 protein complex (Volkmer and Karnitz, 1999; St Onge et al., 1999; Burtelow et al., 2000; Hang and Lieberman, 2000), and the crystal structure of the heterotrimer has been resolved (Doré et al., 2009; Kemp and Sancar, 2009; Sohn and Cho, 2009; Xu et al., 2009). Since a large number of RAD9 activities and protein interactors have been identified, it is clear that RAD9 protein is functionally complex.

Figure 1.

Human RAD9 functional domains and protein–protein interaction sites. Lines correspond to regions of the 391 amino acids long human RAD9 protein that have been assigned functional significance (dotted line) or identified as a site capable of interacting with another protein (solid line). Functional domains: BH3, pro-apoptosis (Komatsu et al., 2000); EXO, 3′–5′ exonuclease activity (Bessho and Sancar, 2000); NLS, nuclear localization signal (Hirai and Wang, 2002). Protein–protein interaction sites: AR, androgen receptor, prostate maintenance (Wang et al., 2004); BCL2, anti-apoptotic protein (Komatsu et al., 2000); BCL-X_L, anti-apoptotic protein (Komatsu et al., 2000); CAD, carbamoyl phosphate synthetase/aspartate transcarbamoylase/dihydroorotase, de novo synthesis of pyrimidine nucleotides and cell growth (Lindsey-Boltz et al., 2004); HUS1, cell cycle checkpoint protein (Doré et al., 2009; Sohn and Cho, 2009; Xu et al., 2009); MLH1, mismatch DNA repair (He et al., 2008); RAD1, cell cycle checkpoint protein (Doré et al., 2009; Sohn and Cho, 2009; Xu et al., 2009); TLK1, tousled-like kinase 1, molecular chaperone in DNA repair (Sunavala-Dossabhoy and De Benedetti, 2009); TOPBP1, topoisomerase IIbeta-binding protein 1, DNA replication and damage response (Mäkiniemi et al., 2001; Greer et al., 2003); TRP2, tetratricopeptide repeat protein 2, steroid receptor chaperoning (Xiang et al., 2001). BCL2 and BCL-X_L bind the BH3 domain. In addition, RAD9 interacts with more proteins and has other functions not indicated because the regions of RAD9 responsible have not been identified.

Evidence of a role for RAD9 in carcinogenesis

Given the roles of RAD9 in growth control and maintaining genomic stability, it is reasonable to think that the protein is important for tumorigenesis. Studies by numerous laboratories have linked aberrations in RAD9 abundance to a variety of cancers or an impact on phenotypes representing hallmark features characteristic of neoplastic transformation. Maniwa et al. (2005) found that 33% (16/48) of non-small cell lung carcinoma tissue samples had cells with aberrantly high levels of RAD9 protein. In another study, Cheng et al. (2005) demonstrated that 52.1% (25/48) of breast tumors overexpressed RAD9 mRNA, which correlated with tumor size and local recurrence. These investigators also showed that MCF-7 breast cancer cells overexpressed RAD9, and reduction in RAD9 protein levels using RNAi inhibited cell proliferation, thus linking RAD9 abundance to cell growth control. A good correlation between overexpression of RAD9 and malignant thyroid neoplasms of follicular cell origin was also reported (Kebebew et al., 2006). Finally, Zhu et al. (2008) found using immunostaining that 45.1% (153/339) of human prostate cancer tissue specimens had high levels of RAD9, while the protein was barely detectable in only 3.8% (2/52) of non-cancerous prostate tissue biopsy controls. There was a strong, statistically significant correlation between RAD9 level and prostate cancer stage. Moreover, these studies demonstrated that prostate cancer cell lines (4/4) have very high levels of the protein, and the ability of siRNA to reduce RAD9 protein abundance correlated strongly with reduced or even eliminated tumorigenicity of the cells when injected into nude mice. This latter study established the functional relationship between RAD9 and tumorigenicity, and in particular that RAD9 can act as an oncogene in the context of prostate cancer. For breast and prostate cancer, gene amplification or aberrant methylation of a transcription repressor region in the second intron of RAD9 can at least in part account for the high levels of the protein observed (Cheng et al., 2005; Zhu et al., 2008).

Human and mouse cells with reduced levels of RAD9 demonstrate a variety of phenotypes, including hypersensitivity to DNA damaging agents, genomic instability, DNA repair deficiency and altered cell cycle checkpoints (Lieberman, 2006). These characteristics indicate that RAD9 is critical for the proper cellular response to DNA damage. Smilenov et al. (2005) found that combined haploinsufficiency for ATM and RAD9 enhance tumorigenesis related cellular characteristics, such as radiation-induced transformation and reduced apoptosis. Consistent with these findings, Hu et al. (2008) reported that mice with a targeted homozygous deletion of RAD9 in keratinocytes are highly susceptible to the development of 7,12-dimethylbenzanthracene-induced skin tumors. Therefore, RAD9 is important for processing damaged DNA and can function as a tumor suppressor for skin tumors, in contrast to the role of the protein as an oncogene in several other cancers.

RAD9 likely plays a direct role in tumorigenesis and does not simply stabilize the genome or promote cell growth for the tumorigenic activity of a second site tumor suppressor or oncogene. This is supported by published studies (Zhu et al. 2008), where non-tumorigenic PWR-1E cells ectopically overexpressing RAD9 form abnormal growths relative to controls. Also, when RAD9 is down-regulated in human prostate cancer DU145 cells, the cells still grow well but are much less tumorigenic, indicating that RAD9 plays an important role in the tumorigenesis process unrelated to promoting cell survival.

Genes with dual cancer roles: oncogene and tumor suppressor

There are several proteins, in addition to RAD9, that can contribute to tumorigenesis when present or active at aberrantly high or low levels, and thus function as oncogenes or tumor suppressors. Caveolin 1 is one example of such a dual function protein, which can act differently depending upon tumor stage and tissue of origin (Williams and Lisanti, 2005). Caveolin 1 levels can be high or low, but are fairly consistent within independent tumors of the same type and tissue. Up-regulation of the Caveolin 1 gene, CAV1, can inhibit mammary epithelial cell transformation and tumorigenesis, as well as metastasis. In contrast, high levels of CAV1 expression in the prostate can promote tumor development and cause metastatic disease. Caveolin 1 has domains that function as growth promoting and inhibiting elements, and the differential activation of those elements, in combination with protein-binding partner interactions in specific tissue environments, are thought to determine whether growth inhibiting or promoting effects will have the greatest biological impact.

Transforming growth factor-beta (TGF-β) can demonstrate tumor-promoting and tumor-suppressing activity (Pardali and Moustakas, 2007). TGF-β is thought to function as a tumor suppressor by inducing cell cycle arrest and apoptosis at an early stage of tumorigenesis, and thus inhibit the initial growth of tumors. In contrast, at later stages in the process, TGF-β can be secreted from cells and promote tumor cell invasiveness as well as metastasis through paracrine and autocrine activities. TGF-β can also regulate angiogenesis and suppress immune surveillance of developing tumors, activities that support tumorigenesis.

E2F1 is a transcription factor that can, in addition, promote or suppress tumorigenesis (Bell and Ryan, 2004). It can regulate cell cycle progression and cause aberrant cell proliferation to promote tumor formation. E2F1 can also act as a pro-apoptotic protein under certain conditions and thus prevent tumorigenesis.

NOTCH1 is another protein that can function as both an oncogene and tumor suppressor. It is an oncogene in prostate cancer by promoting tumor invasion and metastasis (Shou et al., 2001; Bin Hafeez et al., 2009; Wang et al., 2010), but acts as a tumor suppressor in mouse skin cancer. Loss of NOTCH1 in skin results in activation of β-catenin-mediated signaling (Nicolas et al., 2003) and impairs the skin barrier integrity, thus creating a chronic injury/wound-like microenvironment (Demehri et al., 2009). These oncogene-prostate and tumor suppressor-skin relationships in particular are reminiscent of RAD9.

These reports establish that RAD9 is one of several proteins capable of acting as an oncogene or tumor suppressor. The relationships, if any, among members of this group, including RAD9, CAV1, TGF-β, E2F1 and NOTCH1 in the context of carcinogenesis have yet to be defined. However, determining the aforementioned relationships would not only be important from a mechanistic standpoint but would also have the potential for translational impact.

Functions of RAD9 critical for carcinogenesis

It is not clear whether one or more of the previously identified activities of RAD9 are critical for the role of the protein in the development of cancer. More interestingly, the mechanism by which RAD9 can act as an oncogene or tumor suppressor is not established but might in fact be related to several of the protein's activities. For example, overproduction of RAD9 was previously demonstrated to induce transcription of p21 and other downstream target genes (Yin et al., 2004). Therefore, it is possible that high levels of the protein can induce carcinogenesis in the prostate or certain other organs by inappropriately transactivating genes critical for cell growth control and oncogenesis. The situation is even more complicated since overproduction of RAD9 can cause apoptosis in certain cells (Komatsu et al., 2000), and this could be considered tumor suppressive. However, this does not appear to be an issue in the tissues where tumors form when RAD9 levels are high. In contrast, cells with reduced levels of RAD9 exhibit a number of aberrant phenotypes, including genomic instability, decreased homologous recombination repair and mismatch repair abilities, as well as altered cell cycle checkpoint control (Hopkins et al., 2004; Dang et al., 2005; Pandita et al., 2006; He et al., 2008; Zhang et al., 2008). Therefore, it is possible that skin cells null for RAD9 are highly susceptible to genotoxin-induced tumorigenesis because of inherent genomic instability, inability to properly process damaged DNA, and/or improper regulation of cell cycle progression after DNA damage is incurred. To help conceptualize the role of RAD9 in tumorigenesis, the protein can be considered a set of linked functional domains, almost separate proteins fused together. Over or under production of the entire protein might have differential and more domineering effects on one subset as opposed to another of its many functions. Perhaps in the context of the prostate, thyroid, breast or lung, the transactivity function has the most profound impact on carcinogenesis and is activated when the protein is present at high levels. In skin cells, underproduction of RAD9 and its impact on reducing DNA repair capacity, for example, might have the greatest influence on the development of carcinogen-induced tumors. Therefore, RAD9 can be viewed as a complex multi-functional protein with unique, separate domains, and not just a single entity with a simple, one activity portfolio. This model could explain the dual functions of the protein as a tumor suppressor or oncogene when present at different levels in the context of different tissue environments.

Future directions

The finding that proteins such as RAD9 can act as an oncogene or tumor suppressor adds a new level of complexity to understanding how cancer develops and progresses. Investigating the relationship between RAD9 structure and function, the impact of protein-binding partners on RAD9 activity, as well as how the tumor microenvironment might influence RAD9-mediated processes could be exploited to devise novel targeted therapies to treat cancer and possibly other human diseases, by rationally reducing or increasing RAD9 levels. This approach can be considered part of an emerging new strategy to combat cancer, where treatments are custom designed and based on the molecular genetic profile of normal versus cancerous tissues in patients. RAD9 can function in multiple cellular processes, raising special challenges when considering the utility of ultimately targeting the protein level or activity for therapy. Given the multiple roles of RAD9 in the cell, it might under certain circumstances be more advantageous to reduce overall activity, while in other instances therapeutic efficacy might be enhanced if RAD9 activity were increased. For example, increasing levels of the protein could activate apoptosis and thus kill tumor cells when the programmed cell death process is normally inactive. On the other hand, neutralizing RAD9 function could sensitize tumor cells to the killing effects of chemotherapeutic or radiotherapeutic treatment by reducing the ability to repair damaged DNA. As technologies advance, creative strategies might arise to selectively enhance or neutralize the activity of specific protein functional domains or protein–protein interaction sites, as those directly relevant to carcinogenesis are identified, and not simply the entire RAD9 protein. Small molecule inhibitors might be designed, for example, to selectively bind specific catalytic pockets of RAD9 and disable only a subset of the available functions. Protein-binding partners might serve as a more indirect route to knock out specific activities of RAD9. The structure, function and regulatory relationships of RAD9 are complex, but such strategies to manipulate this molecule would be useful to exploit in a well-defined manner to define basic molecular mechanisms of carcinogenesis and for application in the clinic.