Secondary Logo

Journal Logo

Expression Analysis of RbBP6 in human cancers

a Prospective biomarker

Mbita, Zukilea; Hull, Rodneyb; Mbele, Mzwandileb; Makhafola, Tshepisob; Dlamini, Zodwab

doi: 10.1097/CAD.0000000000000809
Review Article
Free

Retinoblastoma binding protein 6 (RBBP6) is a cancer-related protein that has been implicated in the regulation of cell cycle and apoptosis. RBBP6 isoform 1 has been demonstrated to interact with two tumour suppressors, p53 and pRB. Isoform 1 been shown to regulate p53 through its ubiquitin ligase activity, thus implicating in cell cycle regulation and apoptosis. Isoforms 1 and 2 are multidomain proteins containing a domain with no name (DWNN) domain, a Zinc Finger, a RING Finger, an Rb-binding domain and a p53-binding domain. The RBBP6 isoform 3 comprises the DWNN domain only. Isoform 4 lacks the Rb-binding domain but its role is less understood. RBBP6 isoform 3 has been reported as a cell cycle regulator with anticancer potential. There have been several studies that have clearly demonstrated that RBBP6 may be an important biomarker for cancer diagnosis and a potential drug target for cancer treatment. This work focused on differential expression of RBBP6 transcripts in different cancers, providing detailed analysis of their potential as diagnostic biomarkers for different cancers. These cancers include breast, liver, cervical and colon carcinomas. The expression of RBBP6 transcripts may further provide better understanding of the role of the RBBP6 in carcinogenesis and cell homeostasis.

aDepartment of Biochemistry, Microbiology and Biotechnology, University of Limpopo, Sovenga

bSA-MRC/UP Precision Prevention and Novel Drug Targets for HIV-Associated Cancers Extramural Unit, Cancer Research Institute, University of Pretoria, Faculty of Health Sciences, Pretoria, South Africa

Received 12 March 2019 Revised form accepted 21 May 2019

Correspondence to Zukile Mbita, PhD, Department of Biochemistry, Microbiology and Biotechnology, University of Limpopo, Private Bag X1106, Sovenga 0727, South Africa, Tel: +27 0 15 268 3019; fax: +27 015 268 2976; e-mail: zukile.mbita@ul.ac.za

Back to Top | Article Outline

Introduction

Cancer is a major public health problem across the globe. According to GLOBOCAN (Global Burden of Cancer Study), it was estimated that about 14.1 million new cancer cases and 8.2 million deaths occurred in 2012, worldwide. Over the years, the burden has moved to less developed countries, which currently accounts for about 57% of cases and 65% of cancer deaths, worldwide [1]. Lung cancer remains the leading cause of cancer death among males in both more and less developed countries [2]. Although occurrence rates for all cancers combined are approximately twice as high in more developed than in less developed countries in both males and females, mortality rates are only 8–15% higher in more developed countries [3].

Cancers arise through progressive mutations in response to a variety of environmental impacts (viruses, carcinogens, etc.). However, they progress to form disease by avoiding cancer suppression mechanisms (immunity, hypoxia, programmed cell death, etc.). They can do this sometimes by mutation of their DNA, but it is increasingly understood that this is heavily driven by altered gene expression and posttranscriptional regulation (epigenetic modification, alternative splicing, miRNA, etc.).

Bradner et al. [4] and Zanconato et al. [5] have reiterated the importance of transcriptional activation within cancer cells, further implicating the importance of the transcriptional coactivators, Yes–associated protein and the transcriptional coactivator with PDZ-binding domain. Proliferation dysregulation is also a key feature of cancer development. Both cell cycle and apoptosis are linked to proliferation rates and gene-regulated processes. One of the genes that has been heavily implicated in both processes is the RBBP6 gene. RBBP6 gene products have been shown to interact with two of the most studied tumour suppressors, p53 and pRB [6].

Retinoblastoma binding protein 6 (RBBP6) is a 250-kDa splicing nuclear protein, previously implicated in the regulation of cell cycle and apoptosis. Through alternative splicing, the human RBBP6 gene codes for three protein isoforms and isoform 3 consist of the DWNN (domain with no name domain) only while the other two isoforms, 1 and 2, comprise additional zinc, RING, retinoblastoma and p53 binding domains. The RBBP6 gene is localized on chromosome 16p12.2 [7–9]. Currently, it has been documented that there are four protein isoforms of this gene due to different processes that include alternative splicing (mRNA) and alternative use of RBBP6 promoters. One of its isoforms has been reported to interact with two tumour suppressor proteins, retinoblastoma protein (pRb) and p53 [9,10]. These interactions suggest diversity in its function. This gene produces multifunctional gene products that have been implicated in DNA replication [11], development [12], proliferation [13] and metastasis [14,15]. The RBBP6 gene encodes four protein isoforms, 1, 2, 3 and 4 [16]. These isoforms, at least isoforms 1, 2 and 3 are derived from the two major mRNA transcripts, a 1.1- and 6.1-kb transcripts. Isoform 1 is encoded by the 6.1-kb transcript, while alternative splicing of the 6.1-kb transcript results in variant 2, thus producing variants 1 and 2. Isoform 3 encoded by a 1.1-kb transcript is also known as a Domain With No Name (DWNN) [17,18]. It remains a matter of debate how the isoform 4 is derived. Additionally, both isoforms 1 and 3 are almost well understood, while isoforms 2 and 4 remain a mystery. Diversity in RBBP6 gene products may be critical in understanding how this gene influences the carcinogenesis process. Ntwasa [16] briefly reviewed the function of RBBP6, most critically, labelling this interesting gene as another monitor of p53.

Back to Top | Article Outline

Functional diversity of RBBP6 isoforms

There is sufficient evidence that RBBP6 multiple splice variants are involved in different cellular mechanisms and may have opposing functions where RBBP6 isoform 1 may be procarcinogenic while isoform 3 may be anticarcinogenic, depending on the cells they are expressed in. In gastric cancer, RBBP6 has been shown to be expressed in cancer stem cells [15]. Interestingly, we previously showed that the smallest isoform, RBBP6 isoform 3, is involved in cell growth inhibition and cell cycle arrest [17]. These opposing effects further advocate for the opposing functions, at least between RBBP6 isoforms 1 and 3. Most previous studies have shown that the RBBP6 isoform 1 is procarcinogenic while isoform 3 is strongly linked to anticancer activities. RBBP6 isoform 1 has been linked to regulation and stability of p53 and YB1, which further suggests its involvement in tumour progression [19]. It is one of the few proteins that interacts with two major tumour suppressor genes, p53/TP53 and the retinoblastoma gene product (pRb) [18]. Through its RING finger domain, RBBP6 isoform 1 has been reported to mediate ubiquitination of TP53 by Mdm2, an E3 ubiquitin ligase [20]. RBBP6 promotes the degradation of p53, thereby increasing cell proliferation. In mice, the bigger isoform is referred to as PACT, while the rat homologue is the P2P-R [6,9]. These homologues provided the initial understanding of the RBBP6 function. There has been a growing evidence of the role of RBBP6 in cellular processes, but there is a limited distinction on which isoform performs which role. For example, recently, Xiao et al. [21] expertly showed that RBBP6 knockdown sensitized colon cancer cells to radiotherapy. This concurs with several pieces of evidence that has been documented showing that RBBP6 is involved in anticancer activities. One also needs to determine which isoform is involved in anticancer activities between isoforms 2 and 1. Care should be taken when referring to the function of this gene because it encodes a variety of isoforms that may have differing or opposing functions. There has been no study that shows that expression of these variants depends on each other. Table 1 summarizes the different known functions of RBBP6 in different cellular homeostasis processes.

Table 1

Table 1

Back to Top | Article Outline

Regulation of RBBP6 in carcinogenesis

Recently, Varghese et al. [22] showed that RBBP6 may be under the regulation of miR-424, especially in cervical cancer. In their study, they showed that aberrant DNA promoter methylation of the miR-424 resulted in its inactivation. Most importantly, this study showed that the miR-424 interacts with RBBP6 3′-UTR and silences its activity in SiHa cells. According to this report, it is more likely that the low expression of miR-424 in cervical cancer due to its promoter silencing may support the high expression of RBBP6 that has been reported in cervical cancer cells [23]. The mitogen-activated protein kinase signalling pathway has been implicated in promoting cellular proliferation and cell survival in cervical cells [24]. Teng et al. [23] documented that RBBP6 promoted cervical carcinoma proliferation, viability and migration through the JNK signalling pathway. The data show that: (1) RBBP6 is under the miR-424 regulation and the loss of miR-424 expression due to promoter methylation, resulting in RBBP6 upregulation; (2) the involvement of RBBP6 has been shown to be highly expressed in different cancers; and (3) recent implication of RBBP6 in the upregulation of p-JNK, resulting in the activation of JNK signalling pathway that then phosphorylates c-Jun, which increases its transactivation that is associated with cell survival and cell proliferation. Additionally, downregulation of mR-424 has been reported to contribute to angiogenesis [25], inhibit the proliferation of renal cancer cells [26]. As shown in Figure 1, downregulation of miR-424 by its promoter methylation results in the upregulation of RBBP6, which has been shown to activate the JNK pathway.

Fig. 1

Fig. 1

Back to Top | Article Outline

Localization of the RBBP6 transcript 3 in various human cancers

The RBBP6 has been reported to be overexpressed in most cancers. There are conflicting reports on the expression of RBBP6 in human cancers, but there is a growing evidence for upregulation of this gene. Here, we reviewed the expression and implication of RBBP6 in different cancers, at both mRNA (Fig. 2; Table 2) and protein (Fig. 3 and Table 3) levels. There seems to be a difference in the expression patterns between the RBBP6 variant 3 mRNA versus the expression pattern of the two RBBP6 transcripts, 1 and 2. Additionally, DWNN competes with RBBP6 for binding to the core machinery [17,27]. Table 2 and Figure 2 below give a summary on the expression of RBBP6 mRNAs in several human cancer tissues and human cancer cell lines.

Table 2

Table 2

Table 3

Table 3

Fig. 2

Fig. 2

Back to Top | Article Outline

Localization of the RBBP6 isoform 3 in various human cancers

Previously, the localization of DWNN-containing proteins in different human cancers and their normal tissue controls has been established using a purified antihuman DWNN domain antibody [17]. Mostly, the DWNN-containing RBBP6 proteins were found specifically within the cytoplasm of cancer cells within different carcinomas. This is summarized in Table 3 and Figure 3 where localization of the DWNN domain-containing proteins showed differential localization in different cancers, including hepatocellular carcinoma, cervical cancer, breast cancer and ovarian cancer.

Fig. 3

Fig. 3

Fig. 4

Fig. 4

Back to Top | Article Outline

Interaction of RBBP6 and tumour suppressors, pRb and p53

During apoptosis and cell cycle arrest, the cell responds by activating a cascade of genes involved in cell growth. RBBP6, through its pRb- and p53-binding domains, interacts with both p53 and pRB. One of the mouse homologues of RBBP6, P2P-R, is a truncated form of RBBP6, lacking the DWNN that binds p53 and pRB [9]. The role of p53 during cell proliferation is well documented and any increase or decrease in the expression of this protein will have a positive or negative effect on cell survival [28].

There are many known tumour suppressor genes encoding proteins that inhibit the growth of potential cancerous cells by effecting cell cycle regulation and apoptosis. Of these genes, Rb and p53 are the most studied tumour suppressor genes implicated in cancer progression [29,30]. Based on its structure, it was predicted that RBBP6 is a ubiquitin ligase [18], and it was only later that the RBBP6-mediated ubiquitination (Fig. 3) of two cell homeostasis-related proteins, YB-1 [19] and p53 [20], was experimentally established that demonstrated, respectively.

Back to Top | Article Outline

Conclusion

In conclusion, it is clear that RBBP6 is involved in cancer development and is correlated with poor prognosis in various cancers. The frequent upregulation of RBBP6 highlights its potential as a novel therapeutic target for most cancers. RBBP6 may be a target of deregulation in cancer progression, as its expression pattern changes in human cancers. Moreover, alterations of its expression in different cell states also advocate well for its involvement in carcinogenesis and apoptosis. Isoform 1 is upregulated in cancers and promotes cell proliferation and may be considered as antiapoptotic, while isoform 3 is antiproliferative and is downregulated in cancers. The potential clinical value of RPPB6 alone or in combination with p53 and Rb as novel biomarkers in cancers should be investigated extensively.

Back to Top | Article Outline

Acknowledgements

This work was supported by the South African Medical Research Council and the National Research Foundation of South Africa.

Back to Top | Article Outline

Conflicts of interest

There are no conflicts of interest.

Back to Top | Article Outline

References

1. Ferlay J, Steliarova-Foucher E, Lortet-Tieulent J, Rosso S, Coebergh JW, Comber H, et al. Cancer incidence and mortality patterns in Europe: estimates for 40 countries in 2012. Eur J Cancer. 2013; 49:1374–1403
2. Lam WK, White NW, Chan-Yeung MM. Lung cancer epidemiology and risk factors in asia and africa. Int J Tuberc Lung Dis. 2004; 8:1045–1057
3. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J Clin. 2015; 65:87–108
4. Bradner JE, Hnisz D, Young RA. Transcriptional addiction in cancer. Cell. 2017; 168:629–643
5. Zanconato F, Battilana G, Forcato M, Filippi L, Azzolin L, Manfrin A, et al. Transcriptional addiction in cancer cells is mediated by YAP/TAZ through BRD4. Nat Med. 2018; 24:1599–1610
6. Simons A, Melamed-Bessudo C, Wolkowicz R, Sperling J, Sperling R, Eisenbach L, Rotter V. PACT: cloning and characterization of a cellular p53 binding protein that interacts with Rb. Oncogene. 1997; 14:145–155
7. Gao S, Scott RE. P2P-R protein overexpression restricts mitotic progression at prometaphase and promotes mitotic apoptosis. J Cell Physiol. 2002; 193:199–207
8. Gao S, Witte MM, Scott RE. P2P-R protein localizes to the nucleolus of interphase cells and the periphery of chromosomes in mitotic cells which show maximum P2P-R immunoreactivity. J Cell Physiol. 2002; 191:145–154
9. Sakai Y, Saijo M, Coelho K, Kishino T, Niikawa N, Taya Y. Cdna sequence and chromosomal localization of a novel human protein, RBQ-1 (RBBP6), that binds to the retinoblastoma gene product. Genomics. 1995; 30:98–101
10. Witte MM, Scott RE. The proliferation potential protein-related (P2P-R) gene with domains encoding heterogeneous nuclear ribonucleoprotein association and Rb1 binding shows repressed expression during terminal differentiation. Proc Natl Acad Sci U S A. 1997; 94:1212–1217
11. Miotto B, Chibi M, Xie P, Koundrioukoff S, Moolman-Smook H, Pugh D, et al. The RBBP6/ZBTB38/MCM10 axis regulates DNA replication and common fragile site stability. Cell Rep. 2014; 7:575–587
12. Scudiero R, Esposito MG, Simoniello P, Motta CM. Retinoblastoma binding protein 6 and crystallin lambda 1 are cadmium-responsive genes in zebrafish embryos and adults retinae. C R Biol. 2017; 340:197–203
13. Huang P, Ma X, Zhao Y, Miao L. The C. Elegans homolog of RBBP6 (RBPL-1) regulates fertility through controlling cell proliferation in the germline and nutrient synthesis in the intestine. Plos One. 2013; 8:e58736
14. Chen J, Tang H, Wu Z, Zhou C, Jiang T, Xue Y, et al. Overexpression of RBBP6, alone or combined with mutant TP53, is predictive of poor prognosis in colon cancer. Plos One. 2013; 8:e66524
15. Morisaki T, Yashiro M, Kakehashi A, Inagaki A, Kinoshita H, Fukuoka T, et al. Comparative proteomics analysis of gastric cancer stem cells. Plos One. 2014; 9:e110736
16. Ntwasa M. Retinoblastoma binding protein 6, another p53 monitor. Trends Cancer. 2016; 2:635–637
17. Mbita Z, Meyer M, Skepu A, Hosie M, Rees J, Dlamini Z. De-regulation of the RBBP6 isoform 3/DWNN in human cancers. Mol Cell Biochem. 2012; 362:249–262
18. Pugh DJ, Ab E, Faro A, Lutya PT, Hoffmann E, Rees DJ. DWNN, a novel ubiquitin-like domain, implicates RBBP6 in mRNA processing and ubiquitin-like pathways. BMC Struct Biol. 2006; 6:1
19. Chibi M, Meyer M, Skepu A, G Rees DJ, Moolman-Smook JC, Pugh DJ. RBBP6 interacts with multifunctional protein YB-1 through its RING finger domain, leading to ubiquitination and proteosomal degradation of YB-1. J Mol Biol. 2008; 384:908–916
20. Li L, Deng B, Xing G, Teng Y, Tian C, Cheng X, et al. PACT is a negative regulator of p53 and essential for cell growth and embryonic development. Proc Natl Acad Sci U S A. 2007; 104:7951–7956
21. Xiao C, Wang Y, Zheng M, Chen J, Song G, Zhou Z, et al. RBBP6 increases radioresistance and serves as a therapeutic target for preoperative radiotherapy in colorectal cancer. Cancer Sci. 2018; 109:1075–1087
22. Varghese VK, Shukla V, Kabekkodu SP, Pandey D, Satyamoorthy K. DNA methylation regulated micrornas in human cervical cancer. Mol Carcinog. 2018; 57:370–382
23. Teng F, Ruan HJ, Xu J, Ni J, Qian B, Shen R, Gao LJ. RBBP6 promotes human cervical carcinoma malignancy via JNK signaling pathway. Biomed Pharmacother. 2018; 101:399–405
24. Liu B, Fang M, Lu Y, Lu Y, Mills GB, Fan Z. Involvement of JNK-mediated pathway in EGF-mediated protection against paclitaxel-induced apoptosis in siha human cervical cancer cells. Br J Cancer. 2001; 85:303–311
25. Nakashima T, Jinnin M, Etoh T, Fukushima S, Masuguchi S, Maruo K, et al. Down-regulation of mir-424 contributes to the abnormal angiogenesis via MEK1 and cyclin E1 in senile hemangioma: its implications to therapy. Plos One. 2010; 5:e14334
26. Chen B, Duan L, Yin G, Tan J, Jiang X. Simultaneously expressed mir-424 and mir-381 synergistically suppress the proliferation and survival of renal cancer cells—cdc2 activity is up-regulated by targeting WEE1. Clinics (Sao Paulo). 2013; 68:825–833
27. Di Giammartino DC, Li W, Ogami K, Yashinskie JJ, Hoque M, Tian B, Manley JL. RBBP6 isoforms regulate the human polyadenylation machinery and modulate expression of mRNAs with AU-rich 3’ UTRs. Genes Dev. 2014; 28:2248–2260
28. Benedict WF, Xu HJ, Hu SX, Takahashi R. Role of the retinoblastoma gene in the initiation and progression of human cancer. J Clin Invest. 1990; 85:988–993
29. Rusan NM, Peifer M. Original CIN: reviewing roles for APC in chromosome instability. J Cell Biol. 2008; 181:719–726
30. Tommasi S, Pinto R, Pilato B, Paradiso A. Molecular pathways and related target therapies in liver carcinoma. Curr Pharm Des. 2007; 13:3279–3287
31. Thul PJ, Åkesson L, Wiking M, Mahdessian D, Geladaki A, Ait Blal H, et al. A subcellular map of the human proteome. Science. 2017; 356:eaal3321
32. Uhlen M, Oksvold P, Fagerberg L, Lundberg E, Jonasson K, Forsberg M, et al. Towards a knowledge-based human protein atlas. Nat Biotechnol. 2010; 28:1248–1250
33. Gao S, Scott RE. Stable overexpression of specific segments of the P2P-R protein in human MCF-7 cells promotes camptothecin-induced apoptosis. J Cell Physiol. 2003; 197:445–452
Keywords:

apoptosis; cancers; carcinogenesis; cell cycle; retinoblastoma binding protein 6 splice variants

Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.