Multiple molecular interactions redundantly contribute to RB-mediated cell cycle control
© The Author(s) 2017
Received: 22 December 2016
Accepted: 28 February 2017
Published: 14 March 2017
The G1-S phase transition is critical to maintaining proliferative control and preventing carcinogenesis. The retinoblastoma tumor suppressor is a key regulator of this step in the cell cycle.
Here we use a structure–function approach to evaluate the contributions of multiple protein interaction surfaces on pRB towards cell cycle regulation. SAOS2 cell cycle arrest assays showed that disruption of three separate binding surfaces were necessary to inhibit pRB-mediated cell cycle control. Surprisingly, mutation of some interaction surfaces had no effect on their own. Rather, they only contributed to cell cycle arrest in the absence of other pRB dependent arrest functions. Specifically, our data shows that pRB–E2F interactions are competitive with pRB–CDH1 interactions, implying that interchangeable growth arrest functions underlie pRB’s ability to block proliferation. Additionally, disruption of similar cell cycle control mechanisms in genetically modified mutant mice results in ectopic DNA synthesis in the liver.
Our work demonstrates that pRB utilizes a network of mechanisms to prevent cell cycle entry. This has important implications for the use of new CDK4/6 inhibitors that aim to activate this proliferative control network.
KeywordsCell cycle Tumor suppressor Structure–function Retinoblastoma Systems biology
Uninhibited cellular division is a feature of cancer cells. As such, pathways that regulate proliferation are typically disrupted in human cancer . At a molecular level, the cell division cycle is frequently controlled by decisions made in the G1 phase . Once through this phase, the cell is committed to DNA replication and ultimately completion of cell division. The retinoblastoma gene product (pRB) has been shown to be a key regulator of the restriction point that is responsible for controlling S-phase entry . The best known function of pRB is the repression of E2F transcription factor activity . RB performs this function by directly binding the transactivation domain of E2Fs, preventing the recruitment of transcriptional activators to influence gene transcription . In addition, pRB can recruit chromatin regulating enzymes, such as histone deacetylases, to assist in transcriptional repression . This blocks gene expression that is necessary for DNA synthesis and cell cycle entry . In the presence of mitogens cyclin dependent kinases phosphorylate pRB, changing its conformation and releasing E2Fs . Free E2Fs are then able to stimulate transcription and S-phase progression. While this model describes cell cycle entry quite accurately, the role for the same molecular interactions between pRB and E2Fs in cell cycle exit is less clear as pRB dependent arrest can occur much faster than E2F repression .
The minimal interaction domain that mediates stable E2F binding to pRB is the large pocket, and this fragment is also the minimal growth suppressing domain [8, 9]. The large pocket is composed of three regions called A, B, and C . The A and B domains of pRB form the pocket in which the transactivation domain of E2Fs bind [10, 11]. In addition, pRB interacts with a number of chromatin regulators, including HDAC containing complexes, through a well conserved interaction site on the B box of pRB known as the LxCxE binding cleft . This binding site is well defined for its ability to contact the LxCxE motif in viral oncoproteins . Simultaneous interactions between E2Fs, pRB, and chromatin regulators through LxCxE interactions form the basis of active transcriptional repression through E2Fs. The C-terminus of pRB is largely unstructured and serves as a contact point for numerous protein interactions [3, 13]. It is required for stable interaction with E2F-DP dimers , as well as a unique interaction with the marked box domain of E2F1 . Analysis of the large pocket of pRB has contributed to our knowledge of E2Fs in cell cycle control. However, there is little to reconcile how multiple competing protein interactions through this domain contribute to pRB’s overall influence on cell proliferation.
Genetic ablation of RB causes defects in cell proliferation control in tissues and in primary cell culture experiments [16, 17]. However, early studies of pRB-mediated cell cycle regulation exploited the RB null SAOS2 osteosarcoma cell line [8, 9, 18]. RB expression in these cells leads to a robust accumulation of 2 N DNA content, indicating a G1 arrest . These studies looked at a variety of mutant versions of pRB in which strong cancer derived mutations were functionless, but low penetrance RB mutations retained the ability to at least partially restrict cell cycle entry [8, 9, 20, 21]. Surprisingly, the low penetrance mutation R661W was defective for E2F binding, but retained the ability to inhibit cell cycle entry [20–22]. More recently, a number of studies have shown that the R661W mutant can regulate cyclin dependent kinase activity through p27, independent of E2F transcriptional control [7, 23]. Importantly, these studies established that the LxCxE binding cleft and C domains within the large pocket also mediate interactions with the anaphase promoting complex and Skp2 to stabilize p27 expression [7, 24]. Surprisingly, a unified model of how E2F dependent and independent proliferative control mechanisms interact has yet to emerge.
To understand the importance of different protein interaction points in the RB large pocket, targeted mutations to disrupt the LxCxE binding cleft [25–28], the canonical E2F binding site [29, 30], and pRB’s unique interaction with E2F1 in the C-terminus [31, 32], have been generated in mice. Analysis of proliferation in cells and tissues from these mutant animals suggests that individual protein interactions play context specific roles. For example, LxCxE binding cleft mutant mice (called Rb1 L , or Rb1 NF ) are viable with hyper proliferation largely limited to mammary ductal epithelium, that is likely due to unresponsiveness to growth inhibitory signals from TGF-β . Importantly, these mice are not spontaneously cancer prone [27, 34], and they are capable of blocking E2F transcription under a number of physiological circumstances . However, repression of E2F targets is diminished following DNA damage, and the ability of these cells to enter senescence is compromised [35, 36]. Furthermore, mutagen treatment induces cancer in these mice under conditions where E2F repression fails . Disruption of pRB’s unique E2F1 interaction in mice (called Rb1 S ) shows no detectable change in proliferative control in tissues or isolated cells . Lastly, mutational disruption of pRB–E2F interactions in Rb1 G/G mice results in cells with accelerated entry into the cell cycle, but normal cell cycle exit [29, 30]. Remarkably, this mutation does not predispose mice to cancer , however, disruption of this interaction in combination with p27 deficiency deregulates cell cycle arrest functions and these mice are highly cancer prone . This result is also provocative because the cell cycle arrest defects in Rb1 G/G ; p27 deficient compound mutants aren’t found in either single mutant strain alone. These data suggest that pRB dependent cell cycle arrest may depend on a complex network of proliferative control signals such that loss of individual functions have limited effect on their own. This concept is underscored by the fact that no targeted knock in strain recapitulates the complete proliferative control and cancer susceptibility phenotypes of Rb1 −/− mice. In this manuscript, we aimed to eliminate individual binding surfaces in the pRB large pocket to determine the extent that each contributes to cell cycle control alone and in combinations using SAOS2 arrest as a read out. Here, we demonstrate that multiple individual binding surfaces in the large pocket contribute to pRB-mediated cell cycle control in cell culture, and provide proof of principle that this network functions endogenously to regulate DNA replication in the liver.
GST pulldowns and western blotting
C33A cells were transfected with either HA-E2F1-3 (along with DP1), myc tagged CDH1 or pRB expression plasmids under the control of CMV promoters using standard calcium phosphate precipitation techniques. 40 h after transfection cells were washed and collected in GSE buffer (20 mM Tris, pH 7.5, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 5 µg/mL leupeptin, 5 µg/mL aprotinin, 0.1 mM Na3VO4, 0.5 mM NaF, and 1 mM DTT) and frozen at −80 °C. Cell extracts were centrifuged and the supernatant was diluted twofold in low salt GSE (20 mM Tris, pH 7.5, 1.5 mM MgCl2, 0.2 mM EDTA, 25 mM DTT, 0.1% NP-40) and combined with glutathione beads and recombinant fusion proteins. GST-RB large pocket (amino acids 379–928) and GST-HPV-E7 recombinant proteins were expressed and purified as previously described . Beads were then washed twice with low salt GSE, boiled in SDS-sample buffer, resolved by SDS-PAGE, and western blotted. HA-tagged proteins were detected using anti-HA 3F10 (Roche), myc-tagged CDH1 was detected using monoclonal antibody 9E11, and pRB was detected with G3-245 (BD Pharmagen). In order to test pRB stability, cells transfected with CMV expressed pRB were treated with 100 µg/mL cycloheximide for 24 h. Extracts were prepared in GSE buffer every 3 h up to 15 h. Extracts were spun down and western blotted for pRB.
SAOS2 cell cycle arrest assays
SAOS2 cells were transfected and harvested as previously described . Briefly 106 cells were plated in 6 cm dishes and transfected with 0.15 µg of CMV-pRB, 1 µg of CMV-CD20 and 3.85 µg of CMV-β-gal, or 1 µg of CMV-CD20 and 4 µg of CMV-β-gal as a negative control, using X-tremeGENE transfection reagent (Roche). Cells were re-plated onto 10 cm dishes 24 h after transfection, and harvested 48 h later. Cells were then stained with a fluorescein conjugated anti-CD20 antibody to mark successfully transfected cells, as well as with propidium iodide (PI) to determine their DNA content. Flow cytometry was then performed to identify the percentage of CD20 positive cells with 2 N DNA content as a measure of G1. In experiments expressing cell cycle arrest as percent change in G1, arrest data was scaled using CMV-pRB and CMV-β-gal as standards for maximal increase and unchanged G1 content allowing comparisons between different batches of experiments.
Animal housing, dissection and histology
All animals were housed and handled as approved by the Canadian Council on Animal Care. Mice were sacrificed at 8 weeks of age, dissected, and livers were processed for downstream applications. For histology, livers were fixed in formalin for 72 h followed by 72 h in PBS before being stored in 70% ethanol. Livers were then embedded in paraffin and five micron sections were cut and stained with Hematoxylin and Eosin. Images were captured on a Zeiss Axioskop 40 microscope and Spot Flex camera, and nuclear area in the livers was calculated using EyeImage software (Empix Imaging, Mississauga, Ontario, Canada).
Ploidy analysis of adult livers
A small piece of frozen liver was added to buffer A (25 mM Tris pH 7.5, 50 mM KCl, 2 mM MgCl2, 1 mM EDTA, 1 mM PMSF). Tissue was ground on ice with a mechanical tissue grinder. Tissue was then homogenized using a 1 mL dounce homogenizer and tight pestle. Nuclei were centrifuged at 12,000×g, then washed in buffer A and centrifuged. The pellet was then resuspended in Propidium Iodide solution (0.5 mg/mL PI, 0.1% NP-40, 0.1% sodium citrate, 40 µg/mL RNase A in PBS). Samples were then analyzed by flow cytometry using standard methods to quantitate DNA content.
RNA isolation and E2F target gene quantification
RNA from livers was isolated using an RNeasy fibrous tissue kit (Invitrogen). Expression levels of the E2F target genes, Pcna, Ccne1 (cyclin E1), Ccna2 (cyclin A2), Tyms (thymidylate synthase), Mcm3, and Rbl1 (p107), were determined using the Quantigene Plex 2.0 reagent system from Affymetrix (Santa Clara, CA) and a BioPlex200 multiplex analysis system as previously reported . Expression levels were normalized to the expression of β-actin.
BrdU staining of tissue sections
To analyze DNA replication, mice were injected with 200 μL of 16 µg/mL BrdU (Sigma) in their peritoneal cavity 2 h before sacrifice. Livers were then isolated, fixed in formalin, embedded, and sectioned according as above. Sections were deparaffinized and rehydrated using a series of xylene and ethanol washes. The sections were brought to a boil in sodium citrate buffer and then maintained at 95 °C for 10 min. The cooled sections were rinsed in water three times for 5 min, and then rinsed in PBS for 5 min. The sections were blocked in phosphate buffered saline (PBS) supplemented with 2.5% horse serum and 0.3% Triton X-100 for 1 h. The sections were then incubated with anti-BrdU antibodies (BD-Biosciences) in blocking buffer overnight at 4 °C and rinsed in PBS three times for 5 min each time. The slides were incubated with horse anti-mouse immunoglobulin G-fluorescein isothiocyanate (Vector) for 1 h and rinsed in PBS. The slides were then mounted with Vectashield plus DAPI (Vector). Fluorescent images were captured on a Zeiss Axioskop40 microscope and Spot Flex camera and colored using EyeImage software (Empix Imaging, Mississauga, Ontario, Canada), or a similar system.
A cell culture assay demonstrates molecular redundancy of RB functions in proliferative control
A compound mutant mouse model demonstrates molecular redundancy in RB control of DNA replication
Frequency of compound mutant mice
E2f1 −/−; Rb1 G/+ ; Cdkn1b +/− × E2f1 −/−; Rb1 G/+ ; Cdkn1b +/−
E2f1 −/− ; Rb1 +/+ ; Cdkn1b +/+
E2f1 −/− ; Rb1 +/+ ; Cdkn1b +/−
E2f1 −/− ; Rb1 +/+ ; Cdkn1b −/−
E2f1 −/− ; Rb1 G/+ ; Cdkn1b +/+
E2f1 −/− ; Rb1 G/+ ; Cdkn1b +/−
E2f1 −/− ; Rb1 G/+ ; Cdkn1b −/−
E2f1 −/− ; Rb1 G/G ; Cdkn1b +/+
E2f1 −/− ; Rb1 G/G ; Cdkn1b +/−
E2f1 −/− ; Rb1 G/G ; Cdkn1b −/−
We next wanted to determine the effect of our combined mutations on the regulation of pRB functions related to cell cycle control. To accomplish this, RNA was isolated from adult livers to analyze the expression of E2F target gene transcription. Consistent with our previous findings, E2F target gene expression in Rb1 G/G animals is higher than wild-type levels . Interestingly, triple mutant livers show high expression of some of these target genes (Fig. 4e). However, in some cases, E2F target gene expression is unchanged from wild type and this will be discussed later. To directly measure proliferation in livers, 8-week-old animals were injected with BrdU to label nuclei with actively replicating DNA. Livers were dissected, sectioned and stained for BrdU incorporation. This analysis showed that while both Rb1 G/G and Rb1 G/G ; Cdkn1b −/− ; E2f1 −/− livers display increases in the expression of E2F target genes only triple mutant livers displayed increased BrdU incorporation (Fig. 4e, f). Taken together with the increased nuclear area and 8 N DNA content in triple mutant livers, these results suggest that by mutating the general binding site of pRB, and eliminating both p27 and E2F1, we have recapitulated the DNA replication defects associated with conditional deletion of Rb1 in adult livers. These in vivo results also mirror the effects seen in the SAOS2 arrest assays that suggest that no individual protein interaction with pRB accounts for its activity in cell cycle control. Instead, these data indicate that pRB likely sits in the center of a network of regulators that control DNA replication and cell division.
In this manuscript we aimed to further the understanding of pRB-mediated cell cycle control by disrupting pRB-binding interactions in the large pocket to quantitatively account for its arrest mechanisms. This structure–function analysis demonstrated that in order to disrupt the vast majority of pRB’s cell cycle arrest activity, three different binding surfaces needed to be altered. Surprisingly, no single interaction site was indispensible and disruption of some interaction sites had little effect on their own. We used a genetic cross to cripple these three aspects of pRB function endogenously and the combination caused ectopic DNA replication in the liver. This suggests that pRB may interchangeably use different protein interactions to influence cell cycle advancement. Insights and caveats of our study are discussed below.
It is difficult to predict the proliferative control defects of an Rb1 deficient mouse beyond neonatal lethality due to muscle atrophy . Interestingly, chimeric mice composed of a mixture of wild type and Rb1 −/− cells are viable and demonstrate normal tissue cellularity, even in organs where Rb1 −/− cells contribute extensively . This study reveals that livers containing Rb1 −/− hepatocytes display random, large nuclei, similar to our findings in triple mutant livers . In addition, conditional ablation of Rb1 in the livers of adult mice is reported to cause unscheduled DNA replication . The increase in DNA copy number and BrdU incorporation was indicative of a loss of regulation of DNA synthesis . In an effort to model the effects of the RBGSL mutant in vivo, we combined Rb1 G/G animals with p27 and E2F1 deficiency to produce triple mutant animals (Rb1 G/G ; Cdkn1b −/− ; E2f1 −/−). This combination of mutations lead to a very similar DNA replication phenotype in the liver as complete Rb1 deletion. While this is a similar phenotype as conditional deletion of Rb1, by no means does our study elucidate all that pRB pr E2Fs do to block the cell cycle in this or other tissues. We anticipate that viability of triple mutant mice suggests additional pRB dependent cell cycle arrest mechanisms likely remain functional in these animals. Another important consideration in our efforts to model the RBGSL mutant in vivo is that deleting Cdkn1b and E2f1 is not the equivalent to disrupting the binding sites on pRB that regulate them, as these interaction sites may have additional regulatory effects beyond the downstream targets we have chosen. In addition, loss of E2F1 could diminish cell proliferation even when entry into S-phase is deregulated and this could further complicate the interpretation of our analysis of triple mutant livers. Importantly, others have demonstrated that the choice between proliferation and endoreduplication in hepatocytes is determined by opposing effects of activator E2Fs (such as E2F1) and the E2F7 and E2F8 repressors [45, 46]. It is difficult to predict how the triple mutant combination used here would affect the regulation of this network of genes to cause a switch to endoreduplication. Future experiments using Rb1 gene targeted mice carrying a combination of G, S, or L mutations in a single allele will help resolve some of these complexities.
We observed that some individual mutations contributed modestly to proliferative control alone, and more strongly when in combination with other substitutions. We suggest that this may be due in part to the competition between different cell cycle control mechanisms for access to pRB. For example, we demonstrate that E2F3 and CDH1 can compete for the opportunity to interact with pRB, and this is consistent with previous reports of E2F1 and CDH1 competing for pRB . We suggest that CDH1 interactions with pRB are fundamentally different than other pRB interactors that contact the LXCXE binding site simultaneously with E2Fs . Another way to consider redundancy of function through endogenous pRB is a gene targeted mouse model bearing an R654W mutation (the murine equivalent of the low penetrance human mutation R661W). This mutation not only disrupts E2F binding, it also compromised interactions at the LXCXE cleft , potentially illustrating the effects of multiple mutations in a single pRB molecule akin to RBGC in our studies. Fibroblast cells from these mice possess many features of deregulated proliferation seen in Rb1 −/− cells and this mutation is lethal during embryogenesis . However, some aspects of pRB’s role in differentiation and its ability to respond to senescence inducing stimuli and resistance to tumor formation are retained [48, 49], suggesting that simultaneous deficiency by pRB for multiple interactions can reveal a more dramatic phenotype than loss of single interactions. This conclusion is further supported by deregulated cell cycle control and cancer incidence in Rb1 G/G ; Cdkn1b −/− mice , suggesting loss of multiple pRB dependent proliferative control pathways can be dramatically different than loss of a single pathway.
Consistent with multiple interactions needing to be compromised to abrogate cell cycle arrest by pRB, we also note that some mutations tested in this study, such as the M851A, V852A changes (RBS), have no effect on proliferative control in the SAOS2 assay on their own. We suggest that it may represent a latent proliferative control mechanism used by pRB, and there may be others. A long standing puzzle in the RB field has been the existence of proliferative control mechanisms that are mediated by the N-terminus of pRB, outside of the original growth suppressing large pocket domain [50–52]. Recent work has suggested that the N-terminus also plays a role in regulating DNA replication . This may explain the phenotypic difference in proliferative control between Rb1 −/− animals and that of triple mutant Rb1 G/G ; Cdkn1b −/− ; E2f1 −/− animals as the N-terminus is unaffected by our three mutations. There may also be redundancy between N-terminal and large pocket growth arrest mechanisms. Provocatively, there are also low penetrance mutations in human RB1 that target this region of pRB; further suggesting the N-terminus contributes to pRB’s proliferative control and tumor suppressor functions . We think that interchangeability of different pRB functions in proliferative control best explains our data and also encompasses additional work in the field that has previously been difficult to reconcile.
RB dependent proliferative control is functionally inactivated in the vast majority of cancers, this study furthers our understanding of the importance of the various interaction surfaces of pRB and their roles in cell cycle control. In addition, CDK4/6 inhibitors have recently been developed to reactivate the RB-pathway in cancer [54–56]. Understanding the molecular interactions made by pRB and how they influence cell cycle control and tumor suppression is crucial to the proper implementation of these drugs. We expect that the mutational status of both pRB, as well as its regulation of p27 and E2Fs, will play a critical role in the effectiveness of these drugs. We suggest that patients whose tumor cells have pRB activatable p27 will benefit most from CDK4/6 inhibitors.
cyclin dependent kinase
MJT designed and performed cell cycle arrest experiments in SAOS2 cells, carried out protein interaction assays, and wrote the manuscript. MJC performed cell cycle arrest assays and molecular analysis of livers. ST performed histological analyses on livers. DP bred and analysed mouse phenotypes. JC bred and analysed mouse phenotypes. FAD supervised the study and wrote the manuscript. All authors read and approved the final manuscript.
The authors wish to thank colleagues at the London Regional Cancer Program for frequent discussions and encouragement throughout this study.
The authors declare that they have no competing interests.
Availability of data
All data generated or analyzed during this study are included in this published article.
All animal experiments were approved by the animal use subcommittee at Western following CCAC guidelines.
MJT was funded by OGS and MJT, MJC, JC, and ST were members of the CIHR Strategic Training Program in Cancer Research (CaRTT). MJC was a recipient of a CIHR MD/Ph.D. studentship. FAD is the Wolfe Senior Fellow in Tumor Suppressor Genes. This work was funded by an operating Grant from the CIHR (MOP-89765) to FAD.
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