Retinoblastoma protein


The retinoblastoma protein is a tumor suppressor protein that is dysfunctional in several major cancers. One function of Rb is to prevent excessive cell growth by inhibiting cell cycle progression until a cell is ready to divide. When the cell is ready to divide, Rb is phosphorylated to pRb, leading to the inactivation of Rb. This process allows cells to enter into the cell cycle state. It is also a recruiter of several chromatin remodeling enzymes such as methylases and acetylases.
Rb belongs to the pocket protein family, whose members have a pocket for the functional binding of other proteins. Should an oncogenic protein, such as those produced by cells infected by high-risk types of human papillomavirus, bind and inactivate pRb, this can lead to cancer. The RB gene may have been responsible for the evolution of multicellularity in several lineages of life including animals.

Name and genetics

In humans, the protein is encoded by the RB1 gene located on chromosome 13—more specifically, 13q14.1-q14.2. If both alleles of this gene are mutated early in life, the protein is inactivated and results in development of retinoblastoma cancer, hence the name 'Rb'. Retinal cells are not sloughed off or replaced, and are subjected to high levels of mutagenic UV radiation, and thus most pRb knock-outs occur in retinal tissue.
Two forms of retinoblastoma were noticed: a bilateral, familial form and a unilateral, sporadic form. Sufferers of the former were six times more likely to develop other types of cancer later in life. This highlighted the fact that mutated Rb could be inherited and lent support for the two-hit hypothesis. This states that only one working allele of a tumour suppressor gene is necessary for its function, and so both need to be mutated before the cancer phenotype will appear. In the familial form, a mutated allele is inherited along with a normal allele. In this case, should a cell sustain only one mutation in the other RB gene, all Rb in that cell would be ineffective at inhibiting cell cycle progression, allowing cells to divide uncontrollably and eventually become cancerous. Furthermore, as one allele is already mutated in all other somatic cells, the future incidence of cancers in these individuals is observed with linear kinetics. The working allele need not undergo a mutation per se, as loss of heterozygosity is frequently observed in such tumours.
However, in the sporadic form, both alleles would need to sustain a mutation before the cell can become cancerous. This explains why sufferers of sporadic retinoblastoma are not at increased risk of cancers later in life, as both alleles are functional in all their other cells. Future cancer incidence in sporadic Rb cases is observed with polynomial kinetics, not exactly quadratic as expected because the first mutation must arise through normal mechanisms, and then can be duplicated by LOH to result in a tumour progenitor.
RB1 orthologs have also been identified in most mammals for which complete genome data are available.
RB/E2F-family proteins repress transcription.

Structure denotes function

Rb is a multifunctional protein with many binding and phosphorylation sites. Although its common function is seen as binding and repressing E2F targets, Rb is likely a multifunctional protein as it binds to at least 100 other proteins.
Rb has three major structural components: a carboxy-terminus, a "pocket" subunit, and an amino-terminus. Within each subunit, there are a variety of protein binding sites, as well as a total of 15 possible phosphorylation sites. Generally, phosphorylation causes interdomain locking, which changes Rb's conformation and prevents binding to target proteins. Different sites may be phosphorylated at different times, giving rise to many possible conformations and likely many functions/activity levels.

Cell cycle suppression

Rb restricts the cell's ability to replicate DNA by preventing its progression from the G1 to S phase of the cell division cycle. Rb binds and inhibits E2 promoter-binding–protein-dimerization partner dimers, which are transcription factors of the E2F family that push the cell into S phase. By keeping E2F-DP inactivated, RB1 maintains the cell in the G1 phase, preventing progression through the cell cycle and acting as a growth suppressor. The Rb-E2F/DP complex also attracts a histone deacetylase protein to the chromatin, reducing transcription of S phase promoting factors, further suppressing DNA synthesis.

Rb attenuates protein levels of known E2F Targets

Rb has the ability to reversibly inhibit DNA replication through transcriptional repression of DNA replication factors. Rb is able to bind to transcription factors in the E2F family and thereby inhibit their function. When Rb is chronically activated, it leads to the downregulation of the necessary DNA replication factors. Within 72–96 hours of active Rb induction in A2-4 cells, the target DNA replication factor proteins—MCMs, RPA34, DBF4, RFCp37, and RFCp140—all showed decreased levels. Along with decreased levels, there was a simultaneous and expected inhibition of DNA replication in these cells. This process, however, is reversible. Following induced knockout of Rb, cells treated with cisplatin, a DNA-damaging agent, were able to continue proliferating, without cell cycle arrest, suggesting Rb plays an important role in triggering chronic S-phase arrest in response to genotoxic stress.
One such example of E2F-regulated genes repressed by Rb are cyclin E and cyclin A. Both of these cyclins are able to bind to Cdk2 and facilitate entry into the S phase of the cell cycle. Through the repression of expression of cyclin E and cyclin A, Rb is able to inhibit the G1/S transition.

Repression mechanisms of E2Fs

There are at least three distinct mechanisms in which pRb can repress transcription of E2F-regulated promoters. Though these mechanisms are known, it is unclear which are the most important for the control of the cell cycle.
E2Fs are a family of proteins whose binding sites are often found in the promoter regions of genes for cell proliferation or progression of the cell cycle. E2F1 to E2F5 are known to associate with proteins in the pRb-family of proteins while E2F6 and E2F7 are independent of pRb. Broadly, the E2Fs are split into activator E2Fs and repressor E2Fs though their role is more flexible than that on occasion. The activator E2Fs are E2F1, E2F2 and E2F3 while the repressor E2Fs are E2F4, E2F5 and E2F6. Activator E2Fs along with E2F4 bind exclusively to pRb. pRb is able to bind to the activation domain of the activator E2Fs which blocks their activity, repressing transcription of the genes controlled by that E2F-promoter.

Blocking of pre-initiation complex assembly

The preinitiation complex assembles in a stepwise fashion on the promoter of genes to initiate transcription. The TFIID binds to the TATA box in order to begin the assembly of the TFIIA, recruiting other transcription factors and components needed in the PIC. Data suggests that pRb is able to repress transcription by both Rb being recruited to the promoter as well as having a target present in TFIID.
The presence of pRb may change the conformation of the TFIIA/IID complex into a less active version with a decreased binding affinity. pRb can also directly interfere with their association as proteins, preventing TFIIA/IID from forming an active complex.

Modification of chromatin structure

pRb acts as a recruiter that allows for the binding of proteins that alter chromatin structure onto the site E2F-regulated promoters. Access to these E2F-regulated promoters by transcriptional factors is blocked by the formation of nucleosomes and their further packing into chromatin. Nucleosome formation is regulated by post-translational modifications to histone tails. Acetylation leads to the disruption of nucleosome structure. Proteins called histone acetyltransferases are responsible for acetylating histones and thus facilitating the association of transcription factors on DNA promoters. Deacetylation, on the other hand, leads to nucleosome formation and thus makes it more difficult for transcription factors to sit on promoters. Histone deacetylases are the proteins responsible for facilitating nucleosome formation and are therefore associated with transcriptional repressors proteins.
Rb interacts with the histone deacetylases HDAC1 and HDAC3. Rb binds to HDAC1 in its pocket domain in a region that is independent to its E2F-binding site. Rb recruitment of histone deacetylases leads to the repression of genes at E2F-regulated promoters due to nucleosome formation. Some genes activated during the G1/S transition such as cyclin E are repressed by HDAC during early to mid-G1 phase. This suggests that HDAC-assisted repression of cell cycle progression genes is crucial for the ability of Rb to arrest cells in G1. To further add to this point, the HDAC-Rb complex is shown to be disrupted by cyclin D/Cdk4 which levels increase and peak during the late G1 phase.

Senescence induced by Rb

Senescence in cells is a state in which cells are metabolically active but are no longer able to replicate. Rb is an important regulator of senescence in cells and since this prevents proliferation, senescence is an important antitumor mechanism. Rb may occupy E2F-regulated promoters during senescence. For example, Rb was detected on the cyclin A and PCNA promoters in senescent cells.

S-phase arrest

Cells respond to stress in the form of DNA damage, activated oncogenes, or sub-par growing conditions, and can enter a senescence-like state called "premature senescence". This allows the cell to prevent further replication during periods of damaged DNA or general unfavorable conditions. DNA damage in a cell can induce Rb activation. Rb's role in repressing the transcription of cell cycle progression genes leads to the S phase arrest that prevents replication of damaged DNA.

Activation and inactivation

When it is time for a cell to enter S phase, complexes of cyclin-dependent kinases and cyclins phosphorylate Rb to pRb, allowing E2F-DP to dissociate from pRb and become active. When E2F is free it activates factors like cyclins, which push the cell through the cell cycle by activating cyclin-dependent kinases, and a molecule called proliferating cell nuclear antigen, or PCNA, which speeds DNA replication and repair by helping to attach polymerase to DNA.

Inactivation

Since the 1990s, Rb was known to be inactivated via phosphorylation. Until, the prevailing model was that Cyclin D- Cdk 4/6 progressively phosphorylated it from its unphosphorylated to it hyperphosphorylated state. However, it was recently shown that Rb only exists in three states: un-phosphorylated, mono-phosphorylated, and hyper-phosphorylated. Each has a unique cellular function.
Before the development of 2D IEF, only hyper-phosphorylated Rb was distinguishable from all other forms, i.e. un-phosphorylated Rb resembled mono-phosphorylated Rb on immunoblots. As Rb was either in its active “hypo-phosphorylated” state or inactive “hyperphosphorylated” state. However, with 2D IEF, it is now known that Rb is un-phosphorylated in G0 cells and mono-phosphorylated in early G1 cells, prior to hyper-phosphorylation after the restriction point in late G1.

Rb mono phosphorylation

When a cell enters G1, Cyclin D- Cdk4/6 phosphorylates Rb at a single phosphorylation site. No progressive phosphorylation occurs because when HFF cells were exposed to sustained cyclin D- Cdk4/6 activity in early G1, only mono-phosphorylated Rb was detected. Furthermore, triple knockout, p16 addition, and Cdk 4/6 inhibitor addition experiments confirmed that Cyclin D- Cdk 4/6 is the sole phosphorylator of Rb.
Throughout early G1, mono-phosphorylated Rb exists as 14 different isoforms. Together, these isoforms represent the “hypo-phosphorylated” active Rb state that was thought to exist. Each isoform has distinct preferences to associate with different exogenous expressed E2Fs.
A recent report showed that mono-phosphorylation controls Rb's association with other proteins and generates functional distinct forms of Rb. All different mono-phosphorylated Rb isoforms inhibit E2F transcriptional program and are able to arrest cells in G1-phase. Importantly, different mono-phosphorylated forms of RB have distinct transcriptional outputs that are extended beyond E2F regulation.

Hyper-phosphorylation

After a cell passes the restriction point, Cyclin E - Cdk 2 hyper-phosphorylates all mono-phosphorylated isoforms. While the exact mechanism is unknown, one hypothesis is that binding to the C-terminus tail opens the pocket subunit, allowing access to all phosphorylation sites. This process is hysteretic and irreversible, and it is thought accumulation of mono-phosphorylated Rb induces the process. The bistable, switch like behavior of Rb can thus be modeled as a bifurcation point:

Control of Rb function by phosphorylation

Presence of un-phosphorylated Rb drives cell cycle exit and maintains senescence. At the end of mitosis, PP1 dephosphorylates hyper-phosphorylated Rb directly to its un-phosphorylated state. Furthermore, when cycling C2C12 myoblast cells differentiated, only un-phosphorylated Rb was present. Additionally, these cells had a markedly decreased growth rate and concentration of DNA replication factors.
This function of un-phosphorylated Rb gives rise to a hypothesis for the lack of cell cycle control in cancerous cells: Deregulation of Cyclin D - Cdk 4/6 phosphorylates un-phosphorylated Rb in senescent cells to mono-phosphorylated Rb, causing them to enter G1. The mechanism of the switch for Cyclin E activation is not known, but one hypothesis is that it is a metabolic sensor. Mono-phosphorylated Rb induces an increase in metabolism, so the accumulation of mono-phosphorylated Rb in previously G0 cells then causes hyper-phosphorylation and mitotic entry. Since any un-phosphorylated Rb is immediately phosphorylated, the cell is then unable to exit the cell cycle, resulting in continuous division.
DNA damage to G0 cells activates Cyclin D - Cdk 4/6, resulting in mono-phosphorylation of un-phosphorylated Rb. Then, active mono-phosphorylated Rb causes repression of E2F-targeted genes specifically. Therefore, mono-phosphorylated Rb is thought to play an active role in DNA damage response, so that E2F gene repression occurs until the damage is fixed and the cell can pass the restriction point. As a side note, the discovery that damages causes Cyclin D - Cdk 4/6 activation even in G0 cells should be kept in mind when patients are treated with both DNA damaging chemotherapy and Cyclin D - Cdk 4/6 inhibitors.

Activation

During the M-to-G1 transition, pRb is then progressively dephosphorylated by PP1, returning to its growth-suppressive hypophosphorylated state Rb.
Rb family proteins are components of the DREAM complex composed of DP, E2F4/5, RB-like And MuvB. The DREAM complex is assembled in Go/G1 and maintains quiescence by assembling at the promoters of > 800 cell-cycle genes and mediating transcriptional repression. Assembly of DREAM requires DYRK1A dependant phosphorylation of the MuvB core component, Lin52 at Serine28. This mechanism is crucial for recruitment of p130/p107 to the MuvB core and thus DREAM assembly.

Consequences of Rb loss

Consequences of loss of Rb function is dependent on cell type and cell cycle status, as Rb's tumor suppressive role changes depending on the state and current identity of the cell.
In G0 quiescent stem cells, Rb is proposed to maintain G0 arrest although the mechanism remains largely unknown. Loss of Rb leads to exit from quiescence and an increase in the number of cells without loss of cell renewal capacity. In cycling progenitor cells, Rb plays a role at the G1, S, and G2 checkpoints and promotes differentiation. In differentiated cells, which make up the majority of cells in the body and are assumed to be in irreversible G0, Rb maintains both arrest and differentiation.
Loss of Rb therefore exhibits multiple different responses within different cells that ultimately all could result in cancer phenotypes. For cancer initiation, loss of Rb may induce cell cycle re-entry in both quiescent and post-mitotic differentiated cells through dedifferentiation. In cancer progression, loss of Rb decreases the differentiating potential of cycling cells, increases chromosomal instability, prevents induction of cellular senescence, promotes angiogenesis, and increases metastatic potential.
In vivo, it is still not entirely clear how and which cell types cancer initiation occurs with solely loss of Rb, but it is clear that the Rb pathway is altered in large number of human cancers. In mice, loss of Rb is sufficient to initiate tumors of the pituitary and thyroid glands, and mechanisms of initiation for these hyperplasia are currently being investigated.

Non-canonical roles

The classic view of Rb's role as a tumor suppressor and cell cycle regulator developed through research investigating mechanisms of interactions with E2F family member proteins. Yet, more data generated from biochemical experiments and clinical trials reveal other functions of Rb within the cell unrelated to tumor suppression.

Functional hyperphosphorylated Rb

In proliferating cells, certain Rb conformations are resistant to CDK phosphorylation and retain other function throughout cell cycle progression, suggesting not all Rb in the cell are devoted to guarding the G1/S transition.
Studies have also demonstrated that hyperphosphorylated Rb can specifically bind E2F1 and form stable complexes throughout the cell cycle to carry out unique unexplored functions, a surprising contrast from the classical view of Rb releasing E2F factors upon phosphorylation.
In summary, many new findings about Rb's resistance to CDK phosphorylation are emerging in Rb research and shedding light on novel roles of Rb beyond cell cycle regulation.

Genome stability

Rb is able to be localize to sites of DNA breaks during the repair process and assist in non-homologous end joining and homologous recombination through complexing with E2F1. Once at the breaks, Rb is able to recruit regulators of chromatin structure such as the DNA helicase transcription activator BRG1. Rb has been shown to also be able to recruit protein complexes such as condensin and cohesin to assist in the structural maintenance of chromatin.
Such findings suggest that in addition to its tumor suppressive role with E2F, Rb is also distributed throughout the genome to aid in important processes of genome maintenance such as DNA break-repair, DNA replication, chromosome condensation, and heterochromatin formation.

Regulation of metabolism

Rb has also been implicated in regulating metabolism through interactions with components of cellular metabolic pathways. RB1 mutations can cause alterations in metabolism, including reduced mitochondrial respiration, reduced activity in the electron transport chain, and changes in flux of glucose and/or glutamine. Particular forms of Rb have been found to localize to the outer mitochondrial membrane and directly interacts with Bax to promote apoptosis.

As a drug target

Rb Reactivation

While the frequency of alterations of the RB gene is substantial for many human cancer types including as lung, esophageal, and liver, alterations in up-steam regulatory components of Rb such as CDK4 and CDK6 have been the main targets for potential therapeutics to treat cancers with dysregulation in the RB pathway. This focus has resulted in the recent development and FDA clinical approval of three small molecule CDK4/6 inhibitors, Ribociclib, & Abemaciclib ) for the treatment of specific breast cancer subtypes. However, recent clinical studies finding limited efficacy, high toxicity, and acquired resistance of these inhibitors suggests the need to further elucidate mechanisms that influence CDK4/6 activity as well as explore other potential targets downstream in the Rb pathway to reactivate Rb's tumor suppressive functions. Treatment of cancers by CDK4/6 inhibitors depends of the presence of Rb within the cell for therapeutic effect, limiting their usage only to cancers where RB is not mutated and Rb protein levels are not significantly depleted.
Direct Rb reactivation in humans has not been achieved. However, in murine models, novel genetic methods have allowed for in vivo Rb reactivation experiments. Rb loss induced in mice with oncogenic KRAS-driven tumors of lung adenocarcinoma negates the requirement of MAPK signal amplification for progression to carcinoma and promotes loss of lineage commitment as well as accelerate the acquisition of metastatic competency. Reactivation of Rb in these mice rescues the tumors towards a less metastatic state, but does not completely stop tumor growth due to a proposed rewiring of MAPK pathway signaling, which suppresses Rb through a CDK-dependent mechanism.

Pro-apoptotic effects of Rb loss

Besides trying to re-activate the tumor suppressive function of Rb, one other distinct approach to treat dysregulated Rb pathway cancers is to take advantage of certain cellular consequences induced by Rb loss. It has been shown that E2F stimulates expression of pro-apoptotic genes in addition to G1/S transition genes, however, cancer cells have developed defensive signaling pathways that protect themselves from death by deregulated E2F activity. Development of inhibitors of these protective pathways could thus be a synthetically lethal method to kill cancer cells with overactive E2F.
In addition, it has been shown that the pro-apoptotic activity of p53 is restrained by the Rb pathway, such that Rb deficient tumor cells become sensitive to p53 mediated cell death. This opens the door to research of compounds that could activate p53 activity in these cancer cells and induce apoptosis and reduce cell proliferation.

Regeneration

While the loss of a tumor suppressor such as Rb leading to uncontrolled cell proliferation is detrimental in the context of cancer, it may be beneficial to deplete or inhibit suppressive functions of Rb in the context of cellular regeneration. Harvesting the proliferative abilities of cells induced to a controlled “cancer like” state could aid in repairing damaged tissues and delay aging phenotypes. This idea remains to be thoroughly explored as a potential cellular injury and anti-aging treatment.

Cochlea

The retinoblastoma protein is involved in the growth and development of mammalian hair cells of the cochlea, and appears to be related to the cells' inability to regenerate. Embryonic hair cells require Rb, among other important proteins, to exit the cell-cycle and stop dividing, which allows maturation of the auditory system. Once wild-type mammals have reached adulthood, their cochlear hair cells become incapable of proliferation. In studies where the gene for Rb is deleted in mice cochlea, hair cells continue to proliferate in early adulthood. Though this may seem to be a positive development, Rb-knockdown mice tend to develop severe hearing loss due to degeneration of the organ of Corti. For this reason, Rb seems to be instrumental for completing the development of mammalian hair cells and keeping them alive. However, it is clear that without Rb, hair cells have the ability to proliferate, which is why Rb is known as a tumor suppressor. Temporarily and precisely turning off Rb in adult mammals with damaged hair cells may lead to propagation and therefore successful regeneration. Suppressing function of the retinoblastoma protein in the adult rat cochlea has been found to cause proliferation of supporting cells and hair cells. Rb can be downregulated by activating the sonic hedgehog pathway, which phosphorylates the proteins and reduces gene transcription.

Neurons

Disrupting Rb expression in vitro, either by gene deletion or knockdown of Rb short interfering RNA, causes dendrites to branch out farther. In addition, Schwann cells, which provide essential support for the survival of neurons, travel with the neurites, extending farther than normal. The inhibition of Rb supports the continued growth of nerve cells.

Interactions

Rb is known to interact with more than 300 proteins, some of which are listed below:
Several methods for detecting the RB1 gene mutations have been developed including a method that can detect large deletions that correlate with advanced stage retinoblastoma.
.