Loss of CDK4/6 activity in S/G2 phase leads to cell cycle reversal.

In mammalian cells, the decision to proliferate is thought to be irreversibly made at the restriction point of the cell cycle1,2, when mitogen signalling engages a positive feedback loop between cyclin A2/cyclin-dependent kinase 2 (CDK2) and the retinoblastoma protein3-5. Contrary to this textbook model, here we show that the decision to proliferate is actually fully reversible. Instead, we find that all cycling cells will exit the cell cycle in the absence of mitogens unless they make it to mitosis and divide first. This temporal competition between two fates, mitosis and cell cycle exit, arises because cyclin A2/CDK2 activity depends upon CDK4/6 activity throughout the cell cycle, not just in G1 phase. Without mitogens, mitosis is only observed when the half-life of cyclin A2 protein is long enough to sustain CDK2 activity throughout G2/M. Thus, cells are dependent on mitogens and CDK4/6 activity to maintain CDK2 activity and retinoblastoma protein phosphorylation throughout interphase. Consequently, even a 2-h delay in a cell's progression towards mitosis can induce cell cycle exit if mitogen signalling is lost. Our results uncover the molecular mechanism underlying the restriction point phenomenon, reveal an unexpected role for CDK4/6 activity in S and G2 phases and explain the behaviour of all cells following loss of mitogen signalling.

Human embryonic stem cell-derived organoid retinoblastoma reveals a cancerous origin.

Retinoblastoma (Rb) is the most prevalent intraocular malignancy in children, with a worldwide survival rate <30%. We have developed a cancerous model of Rb in retinal organoids derived from genetically engineered human embryonic stem cells (hESCs) with a biallelic mutagenesis of the RB1 gene. These organoid Rbs exhibit properties highly consistent with Rb tumorigenesis, transcriptome, and genome-wide methylation. Single-cell sequencing analysis suggests that Rb originated from ARR3-positive maturing cone precursors during development, which was further validated by immunostaining. Notably, we found that the PI3K-Akt pathway was aberrantly deregulated and its activator spleen tyrosine kinase (SYK) was significantly up-regulated. In addition, SYK inhibitors led to remarkable cell apoptosis in cancerous organoids. In conclusion, we have established an organoid Rb model derived from genetically engineered hESCs in a dish that has enabled us to trace the cell of origin and to test novel candidate therapeutic agents for human Rb, shedding light on the development and therapeutics of other malignancies.

Beyond What Your Retina Can See: Similarities of Retinoblastoma Function between Plants and Animals, from Developmental Processes to Epigenetic Regulation.

The Retinoblastoma protein (pRb) is a key cell cycle regulator conserved in a wide variety of organisms. Experimental analysis of pRb's functions in animals and plants has revealed that this protein participates in cell proliferation and differentiation processes. In addition, pRb in animals and its orthologs in plants (RBR), are part of highly conserved protein complexes which suggest the possibility that analogies exist not only between functions carried out by pRb orthologs themselves, but also in the structure and roles of the protein networks where these proteins are involved. Here, we present examples of pRb/RBR participation in cell cycle control, cell differentiation, and in the regulation of epigenetic changes and chromatin remodeling machinery, highlighting the similarities that exist between the composition of such networks in plants and animals.

Structural basis for recognition of the tumor suppressor protein PTPN14 by the oncoprotein E7 of human papillomavirus.

Human papillomaviruses (HPVs) are causative agents of various diseases associated with cellular hyperproliferation, including cervical cancer, one of the most prevalent tumors in women. E7 is one of the two HPV-encoded oncoproteins and directs recruitment and subsequent degradation of tumor-suppressive proteins such as retinoblastoma protein (pRb) via its LxCxE motif. E7 also triggers tumorigenesis in a pRb-independent pathway through its C-terminal domain, which has yet been largely undetermined, with a lack of structural information in a complex form with a host protein. Herein, we present the crystal structure of the E7 C-terminal domain of HPV18 belonging to the high-risk HPV genotypes bound to the catalytic domain of human nonreceptor-type protein tyrosine phosphatase 14 (PTPN14). They interact directly and potently with each other, with a dissociation constant of 18.2 nM. Ensuing structural analysis revealed the molecular basis of the PTPN14-binding specificity of E7 over other protein tyrosine phosphatases and also led to the identification of PTPN21 as a direct interacting partner of E7. Disruption of HPV18 E7 binding to PTPN14 by structure-based mutagenesis impaired E7's ability to promote keratinocyte proliferation and migration. Likewise, E7 binding-defective PTPN14 was resistant for degradation via proteasome, and it was much more effective than wild-type PTPN14 in attenuating the activity of downstream effectors of Hippo signaling and negatively regulating cell proliferation, migration, and invasion when examined in HPV18-positive HeLa cells. These results therefore demonstrated the significance and therapeutic potential of the intermolecular interaction between HPV E7 and host PTPN14 in HPV-mediated cell transformation and tumorigenesis.

p38γ is essential for cell cycle progression and liver tumorigenesis.

The cell cycle is a tightly regulated process that is controlled by the conserved cyclin-dependent kinase (CDK)-cyclin protein complex1. However, control of the G0-to-G1 transition is not completely understood. Here we demonstrate that p38 MAPK gamma (p38γ) acts as a CDK-like kinase and thus cooperates with CDKs, regulating entry into the cell cycle. p38γ shares high sequence homology, inhibition sensitivity and substrate specificity with CDK family members. In mouse hepatocytes, p38γ induces proliferation after partial hepatectomy by promoting the phosphorylation of retinoblastoma tumour suppressor protein at known CDK target residues. Lack of p38γ or treatment with the p38γ inhibitor pirfenidone protects against the chemically induced formation of liver tumours. Furthermore, biopsies of human hepatocellular carcinoma show high expression of p38γ, suggesting that p38γ could be a therapeutic target in the treatment of this disease.

Cyclin D-Cdk4,6 Drives Cell-Cycle Progression via the Retinoblastoma Protein's C-Terminal Helix.

The cyclin-dependent kinases Cdk4 and Cdk6 form complexes with D-type cyclins to drive cell proliferation. A well-known target of cyclin D-Cdk4,6 is the retinoblastoma protein Rb, which inhibits cell-cycle progression until its inactivation by phosphorylation. However, the role of Rb phosphorylation by cyclin D-Cdk4,6 in cell-cycle progression is unclear because Rb can be phosphorylated by other cyclin-Cdks, and cyclin D-Cdk4,6 has other targets involved in cell division. Here, we show that cyclin D-Cdk4,6 docks one side of an alpha-helix in the Rb C terminus, which is not recognized by cyclins E, A, and B. This helix-based docking mechanism is shared by the p107 and p130 Rb-family members across metazoans. Mutation of the Rb C-terminal helix prevents its phosphorylation, promotes G1 arrest, and enhances Rb's tumor suppressive function. Our work conclusively demonstrates that the cyclin D-Rb interaction drives cell division and expands the diversity of known cyclin-based protein docking mechanisms.

Human U3 protein14a is a novel type ubiquitin ligase that binds RB and promotes RB degradation depending on a leucine-rich region.

The nucleolar protein hUTP14a promotes p53 degradation and possesses an oncogene potential. Here, we report that hUTP14a promotes degradation of tumor suppressor retinoblastoma (RB) protein. Sequences alignment showed that hUTP14a contains the RB-binding PENF motif in its C-terminus. We showed that hUTP14a interacted with RB in vivo and in vitro. Further, hUTP14a promoted polyubiquitination and proteasome-dependent turnover of RB. Importantly, purified Flag-hUTP14a facilitated RB ubiquitination in vitro, demonstrating that hUTP14a is an ubiquitin E3 ligase for RB. A BLAST alignment with hUTP14a does not reveal a RING or HECT domain. To define the conserved domain for E3 ligase activity in hUTP14a, the minimum domain for promoting RB degradation was mapped to residues 61-120 of hUTP14a, in which a leucine-rich region (LRR) LxLxxLL was suggested to be conserved. Flag-hUTP14a (ΔLRR), Flag-hUTP14a-MT1(LxLxxLL to LxLxxAA) and Flag-hUTP14a-MT2(LxLxxLL to AxAxxAA) lost the capability of ubiquitinating RB in vitro, demonstrating that LRR is required for the E3 ligase activity of hUTP14a. Consequently, expression of hUTP14a caused upregulation of E2F1 downstream genes, thus promoting cancer cell proliferation. Taken together, we demonstrate that hUTP14a promotes RB degradation through its E3 ligase activity and suggest that the LRR could be a potential conserved E3 ligase domain.

Short linear motif core and flanking regions modulate retinoblastoma protein binding affinity and specificity.

Pocket proteins retinoblastoma (pRb), p107 and p130 are negative regulators of cellular proliferation and multifunctional proteins regulating development, differentiation and chromatin structure. The retinoblastoma protein is a potent tumor suppressor mutated in a wide range of human cancers, and oncogenic viruses often interfere with cell cycle regulation by inactivating pRb. The LxCxE and pRb AB groove short linear motifs (SLiMs) are key to many pocket protein mediated interactions including host and viral partners. A review of available experimental evidence reveals that several core residues composing each motif instance are determinants for binding. In the LxCxE motif, a fourth hydrophobic position that might allow variable spacing is required for binding. In both motifs, flanking regions including charged stretches and phosphorylation sites can fine-tune the binding affinity and specificity of pocket protein SLiM-mediated interactions. Flanking regions can modulate pocket protein binding specificity, or tune the high affinity interactions of viral proteins that hijack the pRb network. The location of SLiMs within intrinsically disordered regions allows faster evolutionary rates that enable viruses to acquire a functional variant of the core motif by convergent evolution, and subsequently test numerous combinations of flanking regions towards maximizing interaction specificity and affinity. This knowledge can guide future efforts directed at the design of peptide-based compounds that can target pocket proteins to regulate the G1/S cell cycle checkpoint or impair viral mediated pRb inactivation.

Conservation and divergence of C-terminal domain structure in the retinoblastoma protein family.

The retinoblastoma protein (Rb) and the homologous pocket proteins p107 and p130 negatively regulate cell proliferation by binding and inhibiting members of the E2F transcription factor family. The structural features that distinguish Rb from other pocket proteins have been unclear but are critical for understanding their functional diversity and determining why Rb has unique tumor suppressor activities. We describe here important differences in how the Rb and p107 C-terminal domains (CTDs) associate with the coiled-coil and marked-box domains (CMs) of E2Fs. We find that although CTD-CM binding is conserved across protein families, Rb and p107 CTDs show clear preferences for different E2Fs. A crystal structure of the p107 CTD bound to E2F5 and its dimer partner DP1 reveals the molecular basis for pocket protein-E2F binding specificity and how cyclin-dependent kinases differentially regulate pocket proteins through CTD phosphorylation. Our structural and biochemical data together with phylogenetic analyses of Rb and E2F proteins support the conclusion that Rb evolved specific structural motifs that confer its unique capacity to bind with high affinity those E2Fs that are the most potent activators of the cell cycle.

The N-Terminal Phosphorylation of RB by p38 Bypasses Its Inactivation by CDKs and Prevents Proliferation in Cancer Cells.

Control of the G1/S phase transition by the Retinoblastoma (RB) tumor suppressor is critical for the proliferation of normal cells in tissues, and its inactivation is one of the most fundamental events leading to cancer. Cyclin-dependent kinase (CDK) phosphorylation inactivates RB to promote cell cycle-regulated gene expression. Here we show that, upon stress, the p38 stress-activated protein kinase (SAPK) maximizes cell survival by downregulating E2F gene expression through the targeting of RB. RB undergoes selective phosphorylation by p38 in its N terminus; these phosphorylations render RB insensitive to the inactivation by CDKs. p38 phosphorylation of RB increases its affinity toward the E2F transcription factor, represses gene expression, and delays cell-cycle progression. Remarkably, introduction of a RB phosphomimetic mutant in cancer cells reduces colony formation and decreases their proliferative and tumorigenic potential in mice.

Structural Conservation and E2F Binding Specificity within the Retinoblastoma Pocket Protein Family.

The human pocket proteins retinoblastoma (Rb), p107, and p130 are critical negative regulators of the cell cycle and contribute to tumor suppression. While strong structural conservation within the pocket protein family provides for some functional redundancy, important differences have been observed and may underlie the reason that Rb is a uniquely potent tumor suppressor. It has been proposed that distinct pocket protein activities are mediated by their different E2F transcription factor binding partners. In humans, Rb binds E2F1-E2F5, whereas p107 and p130 almost exclusively associate with E2F4 and E2F5. To identify the molecular determinants of this specificity, we compared the crystal structures of Rb and p107 pocket domains and identified several key residues that contribute to E2F selectivity in the pocket family. Mutation of these residues in p107 to match the analogous residue in Rb results in an increase in affinity for E2F1 and E2F2 and an increase in the ability of p107 to inhibit E2F2 transactivation. Additionally, we investigated how phosphorylation by Cyclin-dependent kinase on distinct residues regulates p107 affinity for the E2F4 transactivation domain. We found that phosphorylation of residues S650 and S975 weakens the E2F4 transactivation domain binding. Our data reveal molecular features of pocket proteins that are responsible for their similarities and differences in function and regulation.

A Strategy for Direct Chemical Activation of the Retinoblastoma Protein.

The retinoblastoma (Rb) tumor suppressor protein negatively regulates cell proliferation by binding and inhibiting E2F transcription factors. Rb inactivation occurs in cancer cells upon cyclin-dependent kinase (Cdk) phosphorylation, which induces E2F release and activation of cell cycle genes. We present a strategy for activating phosphorylated Rb with molecules that bind Rb directly and enhance affinity for E2F. We developed a fluorescence polarization assay that can detect the effect of exogenous compounds on modulating affinity of Rb for the E2F transactivation domain. We found that a peptide capable of disrupting the compact inactive Rb conformation increases affinity of the repressive Rb-E2F complex. Our results demonstrate the feasibility of discovering novel molecules that target the cell cycle and proliferation through directly targeting Rb rather than upstream kinase activity.

The N Terminus of the Retinoblastoma Protein Inhibits DNA Replication via a Bipartite Mechanism Disrupted in Partially Penetrant Retinoblastomas.

The N-terminal domain of the retinoblastoma (Rb) tumor suppressor protein (RbN) harbors in-frame exon deletions in partially penetrant hereditary retinoblastomas and is known to impair cell growth and tumorigenesis. However, how such RbN deletions contribute to Rb tumor- and growth-suppressive functions is unknown. Here we establish that RbN directly inhibits DNA replication initiation and elongation using a bipartite mechanism involving N-terminal exons lost in cancer. Specifically, Rb exon 7 is necessary and sufficient to target and inhibit the replicative CMG helicase, resulting in the accumulation of inactive CMGs on chromatin. An independent N-terminal loop domain, which forms a projection, specifically blocks DNA polymerase α (Pol-α) and Ctf4 recruitment without affecting DNA polymerases ε and δ or the CMG helicase. Individual disruption of exon 7 or the projection in RbN or Rb, as occurs in inherited cancers, partially impairs the ability of Rb/RbN to inhibit DNA replication and block G1-to-S cell cycle transit. However, their combined loss abolishes these functions of Rb. Thus, Rb growth-suppressive functions include its ability to block replicative complexes via bipartite, independent, and additive N-terminal domains. The partial loss of replication, CMG, or Pol-α control provides a potential molecular explanation for how N-terminal Rb loss-of-function deletions contribute to the etiology of partially penetrant retinoblastomas.

Rapid tumor induction in zebrafish by TALEN-mediated somatic inactivation of the retinoblastoma1 tumor suppressor rb1.

Investigating the in vivo role of tumor suppressor genes in cancer is technically challenging due to their essential requirement during early animal development. To address this bottleneck, we generated genetic mosaic adult zebrafish using TALEN genome editing and demonstrate somatic inactivation of the tumor suppressor retinoblastoma1 (rb1) induces tumorigenesis at high frequency. 11-33% of 1-cell stage embryos injected with TALEN mRNAs targeting rb1 exon 2 or 3 develop tumors beginning as early as 3.5 months of age. Lesions predominantly arise in the brain and show features of neuroectodermal-like and glial-like tumors. Mutant allele analysis is consistent with tumor initiation due to somatic inactivation of rb1, revealing a conserved role for rb1 in tumor suppression across vertebrates. In contrast to genetic mosaics, heterozygous rb1-/+ adults show no evidence of neoplasia, while homozygous mutant rb1-/- are larval lethal. This is the first demonstration that somatic inactivation of a tumor suppressor causes cancer in zebrafish, and highlights the utility of site-specific nucleases to create genetic mosaic zebrafish for tumor suppressor gene discovery. Somatic inactivation with site-directed nucleases in zebrafish presents a rapid and scalable strategy to study tumor suppressor gene function in cancer.

Molecular Determinants for the Inactivation of the Retinoblastoma Tumor Suppressor by the Viral Cyclin-dependent Kinase UL97.

The retinoblastoma (Rb) tumor suppressor restricts cell cycle progression by repressing E2F-responsive transcription. Cellular cyclin-dependent kinase (CDK)-mediated Rb inactivation through phosphorylation disrupts Rb-E2F complexes, stimulating transcription. The human cytomegalovirus (HCMV) UL97 protein is a viral CDK (v-CDK) that phosphorylates Rb. Here we show that UL97 phosphorylates 11 of the 16 consensus CDK sites in Rb. A cleft within Rb accommodates peptides with the amino acid sequence LXCXE. UL97 contains three such motifs. We determined that the first LXCXE motif (L1) of UL97 and the Rb cleft enhance UL97-mediated Rb phosphorylation. A UL97 mutant with a non-functional L1 motif (UL97-L1m) displayed significantly reduced Rb phosphorylation at multiple sites. Curiously, however, it efficiently disrupted Rb-E2F complexes but failed to relieve Rb-mediated repression of E2F reporter constructs. The HCMV immediate early 1 protein cooperated with UL97-L1m to inactivate Rb in transfection assays, likely indicating that cells infected with a UL97-L1m mutant virus show no defects in growth or E2F-responsive gene expression because of redundant viral mechanisms to inactivate Rb. Our data suggest that UL97 possesses a mechanism to elicit E2F-dependent gene expression distinct from disruption of Rb-E2F complexes and dependent upon both the L1 motif of UL97 and the cleft region of Rb.

Role of adenovirus-mediated retinoblastoma 94 in the treatment of human non-small cell lung cancer.

Non­small cell lung cancer (NSCLC) remains the leading cause of cancer­related mortality despite the fact that great advances have been made in therapeutic treatment methods. Therefore, in the present study, the role of adenovirus-mediated retinoblastoma 94 (Ad­RB94) gene therapy in NSCLC was investigated. Following treatment with Ad­RB94, the proportion of A549 cells in the G2/M phase was increased. In the mouse xenograft model, the overexpression of RB94 inhibited the tumor growth compared with the control group and the Ad-­LacZ-treated group. In the transplanted tumors, the overexpression of RB94 induced the apoptosis of tumors as well as an increase in the mRNA levels of cyclinB1. In conclusion, the results of the present study suggested that RB94 may effectively inhibit NSCLC tumor cell growth by inducing G2/M cell cycle arrest and apoptosis, indicating that RB94 may be a promising candidate for adjuvant therapy with radiation or chemotherapy in NSCLC.

PP2A: more than a reset switch to activate pRB proteins during the cell cycle and in response to signaling cues.

In their active hypophosphorylated state, members of the retinoblastoma family of pocket proteins negatively regulate cell cycle progression at least in part by repressing expression of E2F-dependent genes. Mitogen-dependent activation of G1 and G1/S Cyclin Dependent Kinases (CDKs) results in coordinated hyperphosphorylation and inactivation of these proteins, which no longer bind and repress E2Fs. S and G2/M CDKs maintain pocket protein hyperphosphorylated through the end of mitosis. The inactivating action of inducible CDKs is opposed by the Ser/Thr protein phosphatases PP2A and PP1. Various trimeric PP2A holoenzymes have been implicated in dephosphorylation of pocket proteins in response to specific cellular signals and stresses or as part of an equilibrium with CDKs throughout the cell cycle. PP1 has specifically been implicated in dephosphorylation of pRB in late mitosis and early G1. This review is particularly focused on the emerging role of PP2A as a major hub for integration of growth suppressor signals that require rapid inactivation of pocket proteins. Of note, activation of particular PP2A holoenzymes triggers differential activation of pocket proteins in the presence of active CDKs.

The high-risk HPV16 E7 oncoprotein mediates interaction between the transcriptional coactivator CBP and the retinoblastoma protein pRb.

The oncoprotein E7 from human papillomavirus (HPV) strains that confer high cancer risk mediates cell transformation by deregulating host cellular processes and activating viral gene expression through recruitment of cellular proteins such as the retinoblastoma protein (pRb) and the cyclic-AMP response element binding binding protein (CBP) and its paralog p300. Here we show that the intrinsically disordered N-terminal region of E7 from high-risk HPV16 binds the TAZ2 domain of CBP with greater affinity than E7 from low-risk HPV6b. HPV E7 and the tumor suppressor p53 compete for binding to TAZ2. The TAZ2 binding site in E7 overlaps the LxCxE motif that is crucial for interaction with pRb. While TAZ2 and pRb compete for binding to a monomeric E7 polypeptide, the full-length E7 dimer mediates an interaction between TAZ2 and pRb by promoting formation of a ternary complex. Cell-based assays show that expression of full-length HPV16 E7 promotes increased pRb acetylation and that this response depends both on the presence of CBP/p300 and on the ability of E7 to form a dimer. These observations suggest a model for the oncogenic effect of high-risk HPV16 E7. The disordered region of one E7 molecule in the homodimer interacts with the pocket domain of pRb, while the same region of the other E7 molecule binds the TAZ2 domain of CBP/p300. Through its ability to dimerize, E7 recruits CBP/p300 and pRb into a ternary complex, bringing the histone acetyltransferase domain of CBP/p300 into proximity to pRb and promoting acetylation, leading to disruption of cell cycle control.

Lysine methylation-dependent binding of 53BP1 to the pRb tumor suppressor.

The retinoblastoma tumor suppressor protein pRb is a key regulator of cell cycle progression and mediator of the DNA damage response. Lysine methylation at K810, which occurs within a critical Cdk phosphorylation motif, holds pRb in the hypophosphorylated growth-suppressing state. We show here that methyl K810 is read by the tandem tudor domain containing tumor protein p53 binding protein 1 (53BP1). Structural elucidation of 53BP1 in complex with a methylated K810 pRb peptide emphasized the role of the 53BP1 tandem tudor domain in recognition of the methylated lysine and surrounding residues. Significantly, binding of 53BP1 to methyl K810 occurs on E2 promoter binding factor target genes and allows pRb activity to be effectively integrated with the DNA damage response. Our results widen the repertoire of cellular targets for 53BP1 and suggest a previously unidentified role for 53BP1 in regulating pRb tumor suppressor activity.

Retinoblastoma (RB1) pocket domain mutations and promoter hyper-methylation in head and neck cancer.

BACKGROUND: The RB1 gene plays a pivotal role in cell cycle regulation. In this case-control study we searched for alterations in the RB1 pocket domain and its promoter region in head and neck cancer (HNC) patients in the Pakistani population. METHODS: For germline mutation analyses, 380 blood samples from HNC patients and 350 blood samples from control individuals were included. Polymerase chain reaction (PCR) and single strand conformational polymorphism (SSCP) assays, followed by sequence analyses, were used for the RB1 pocket domain mutation screens. For the RB1 promoter methylation screens, 72 HNC tumor samples along with adjacent uninvolved tissues were tested using a methylation-specific polymerase chain reaction (MSP) assay. RESULTS: RB1 (pocket domain and spacer region) sequence analysis revealed one frameshift and seven non-synonymous missense mutations. The frequency of missense mutations in exon 14, i.e., g76474C > T, g76475G > C and g76476A > G, resulting in Arg455Ser, was found to be highest (0.10). Missense mutations g76467G > C (exon14), g76468T > C (exon14), g77041A > T and g77043A > T (exon 16), when analyzed via Alamut biosoftware version 2.0, were found to be present in highly conserved amino acids with Align GVGD scores C15 (GV: 0.00-GD: 21.82), C65 (GV: 0.00-GD: 83.33) and C65 (GV: 0.00-GD: 98.69), respectively. These missense mutations were found to be deleterious by SIFT score: 0.00 (median 3.64). RB1 promoter methylation analysis revealed that 16% of its cytosines (3% in CpG) were methylated in the HNC tumor samples. CONCLUSION: Our findings indicate that both genetic and epigenetic RB1 changes may contribute to the pathogenesis of HNC in the Pakistani population.