RBX1/ROC1-SCF E3 ubiquitin ligase is required for mouse embryogenesis and cancer cell survival
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RBX1 (also known as ROC1) is a RING subunit of SCF (S kp1, C ullins, F-box proteins) E3 ubiquitin ligases, required for SCF to direct a timely degradation of diverse substrates, thereby regulating numerous cellular processes under both physiological and pathological conditions. Previous studies have shown that RBX1 is essential for growth in yeast, Caenorhabditis elegans and Drosophila. The role of RBX1 in mouse development and in regulation of cancer cell survival was unknown. Our recent work demonstrated that RBX1 is an essential gene for mouse embryogenesis, and targeted disruption of RBX1 causes embryonic lethality at E7.5 due to hypoproliferation as a result of p27 accumulation. We also showed that RBX1 is overexpressed in a number of human cancers, and siRNA silencing of RBX1 caused cancer cell death as a result of sequential induction of G2-M arrest, senescence and apoptosis. These findings reveal a physiological role of RBX1 during mouse development and a pathological role for the survival of human cancer cells. Differential outcomes between normal (growth arrest) and cancer cells (cell death) upon RBX1 disruption/silencing suggest RBX1 as a valid anticancer target.
Tan M, Davis SW, Saunders TL, Zhu Y, Sun Y. RBX1/ROC1 disruption results in early embryonic lethality due to proliferation failure, partially rescued by simultaneous loss of p27. Proc Natl Acad Sci USA. 2009; 106:6203–6208
Jia L, Soengas MS, Sun Y. ROC1/RBX1 E3 ubiquitin ligase silencing suppresses tumor cell growth via sequential induction of G2-M arrest, apoptosis, and senescence. Cancer Res. 2009; 69:4974–82
KeywordsSequential Induction Mouse Development Mouse Embryogenesis Early Embryonic Lethality Numerous Cellular Process
RBX1-Cullins E3 ubiquitin ligases and their substrates in mammals
e.g. p21, p27, p57, Cyclins A/D/E, E2F1, Cdc25A/B, PDCC4, FOXO1, Myc, p53, c-Jun, Notch 1/4, IκB, β-Catenin, Orc1, and many more. For near complete list, see cited references
e.g. HIF-α, TEL-JAK2
e.g. MEI-1, Dishevelled (Dsh), Nrf2, RhoBTB2, topoisomeraseI-DNA complex, and caspase 8
e.g. p53, TSC2, Cdt1, c-Jun and Merlin
RBX1/Cullin-5/elongin BC/BC-box proteins/SOCS
e.g. Disabled-1 (Dab1)
e.g. Insulin receptor substrate 1 (IRS-1)
RBX1 in development
RBX1-cullin complexes control the proteolysis of numerous substrates related to cell cycle progression, cell growth and cell death, suggesting that RBX1 may play an important role in development. Indeed, RBX1 is an essential gene in a number of species. In yeast, deletion of Hrt1, the yeast homologue of RBX1, via genetic manipulation, causes yeast death, which can be rescued by human RBX1 or RBX2/SAG [6, 7, 8]. In Caenorhabditis elegans, RBX1 is also crucial for cell cycle progression and chromosome metabolism, as evidenced by severe defects in meiosis, mitotic chromosomal condensation and segregation, and cytokinesis upon siRNA knockdown . In Drosophila, ROC1a, the drosophila homologue of RBX1 is required for cell proliferation and embryo development, and deletion of ROC1a results in animal death . In mouse, the level of RBX1 mRNA was regulated during embryonic development with the strongest expression at embryonic day 7 (E7), followed by a progressive decrease . However, the physiological role of RBX1 in mouse development has not been previously characterized.
Functional characterization using various model systems from yeast to mouse clearly demonstrated that RBX1 is an essential gene for growth and development. Interestingly, some similarity and difference exist between the species with more than one family member of RBX1. For example, death phenotype induced by disruption of ROC1a in Drosophila or of RBX1 in mouse in the presence of their family member ROC2 or RBX2 [10, 12, 13] clearly indicated that these two RBX1 family members are not functionally redundant and are likely to target different sets of substrates during embryonic development. On the other hand, the mechanisms responsible for reduced proliferation as a result of ROC1a/RBX1 disruption seem different between Drosophila and mouse. In Drosophila, disruption of ROC1a, causes lethality due to proliferation failure as a result of accumulation of Ci (a Drosophila ortholog of mouse Gli2), a transcription factor that regulates Hedgehog signaling . Whereas in mouse, disruption of RBX1 does not affect the levels of Gli2, but causes p27 accumulation to suppress proliferation . Furthermore, although RBX1-p27 double null embryos at E9.5 are smaller than wild type littermates, no enhanced apoptosis was detected using the TUNEL assay (unpublished data). Thus, during mouse embryogenesis, RBX1 disruption appears not to induce apoptosis. These observations in normal tissues are strikely different from those seen in human cancer cells (see below) in which RBX1 silencing induces significant levels of apoptosis and senescence.
RBX1 in human cancer cell survival
RBX1-SCF E3 ubiquitin ligases regulate numerous cellular processes. It is not surprising that their dysfunction is associated with a variety of diseases including cancer . For example, an oncogenic F-box protein Skp2, which promotes p27 degradation, is overexpressed in a number of human cancers , whereas a tumor suppressive F-box protein FBW7, which promotes the degradation of several proto-oncogenes, including c-Jun, c-Myc, cyclin E and mTOR undergoes numerous cancer-associated mutations . To define potential roles of RBX1 in human cancers, we recently measured expression of RBX1 in human primary cancer tissues and in cancer cell lines with different tissue origins. We found that RBX1 is overexpressed in a number of human primary cancer tissues, including carcinoma of lung, liver, breast, colon, and ovary, and in many cancer cell lines . We then determined potential biological consequences of reducing RBX1 levels via siRNA silencing. Significantly, RBX1 knockdown inhibited the growth of several human cancer cell lines by sequential induction of G2-M arrest, senescence and apoptosis. Further characterization revealed that G2-M arrest is associated with accumulation of 14-3-3σ and down-regulation of cyclin B1 and Cdc2, whereas apoptosis is associated with modest accumulation of PUMA and significant reduction of Bcl-2, Mcl-1, and survivin. Interestingly, senescence is p53/p21- and p16/pRB-independent . Recently a shRNA library-based functional genomic screen also identified RBX1 as a growth essential gene in a number of human cancer cell lines, although no characterization was further pursued .
Mechanistic studies revealed that RBX1 silencing triggers DNA damage response at the early stage, as demonstrated by induced phosphoralytion of H2AX, Chk1 and Chk2  (and unpublished data), which eventually leads to G2-M arrest, followed by apoptosis and senescence. We hypothesize that either or both sets of RBX1 substrates, which start to accumulate upon RBX1 silencing, are likely involved in the process, leading to phenotypic changes. The first set includes oncogenes (e.g. c-Myc, c-Jun, cyclin E/D), since oncogene activation triggers DNA damage response to induce senescence and apoptosis under certain circumstances [19, 20]. The second set of RBX1-cullin substrates could be DNA replication proteins, such as Orc-1 and Cdt-1, since the accumulation of DNA replication proteins (e.g. Cdt-1) induces DNA rereplication stress and triggers DNA damage  (Fig 2B). Our laboratory is currently testing the hypothesis to further elucidate the mechanism(s) by which RBX1 silencing induces cancer cell killing via induction of G2-M arrest, senescence and apoptosis.
It is rather clear that the mechanism responsible for early embryonic lethality upon RBX1 disruption is quite different from that responsible for cancer cell killing upon RBX1 silencing, although it is not a typically paired comparison. Nevertheless, in mouse, RBX1 knockout leads to p27 accumulation, reduced proliferation and prolonged G1 arrest, whereas in human cancer cells, p27 accumulation and G1 arrest were not observed upon RBX1 silencing . Future studies should be directed to determine if the altered DNA damage response commonly seen in cancer cells after RBX1 silencing can also be observed in RBX1-p27 double null embryos and if so, its contribution to the embryonic lethality in the later stages of development (Fig 2, crosstalk between panel A and B).
The findings from our laboratory demonstrated that RBX1 is an essential gene not only for mouse development but also for human cancer cell survival. The fact that RBX1 is overexpressed in a number of human cancers suggests that abnormal regulation of RBX1 is involved either in human carcinogenesis or in the maintenance of the cancer cell phenotype. Differential response to RBX1 disruption/silencing between normal tissues (reduced proliferation, but no induction of apoptosis during mouse embryogenesis) and cancer cells (enhanced cell killing) may provide a reasonable therapeutic window for cancer cell-specific killing via RBX1 targeting. Thus, future development of siRNA-based therapy by RBX1 silencing or small molecule inhibitors against RBX1 E3 ubiquitin ligases may hold great promise for the treatment of human cancer [22, 23, 24].
This work is supported by the National Cancer Institute grants CA111554 and CA116982 to YS.
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