Rb Families Research Articles

Clinical characteristics and germline mutation spectrum of RB1 in Chinese patients with retinoblastoma: A dual-center study of 145 patients

Retinoblastoma (Rb) is the most common primary intraocular childhood malignancy and one of the main causes of blindness in children. In China, most tumors are diagnosed at an advanced stage and have relatively poor outcomes compared to developed countries. Here, we aimed to update the clinical manifestations and RB transcriptional corepressor 1 (RB1) mutation spectrum in Chinese Rb patients. Medical charts of 184 eyes in 145 Chinese Rb patients belonging to unrelated families were reviewed. Genomic DNA was isolated from peripheral blood of the patients and their parents. Mutation analysis of whole coding regions, promoter regions and flanking splice sites in the RB1 gene was performed. In addition, multiplex ligation-dependent probe amplification (MLPA) was done to detect gross aberrations. Germline RB1 mutations were observed in 37.2% (54/145) of Rb patients. RB1-mutated patients presented with earlier age of diagnosis (p = 0.019), with a significantly larger proportion of bilateral cases (p = <0.001) and secondary malignancies (p = 0.027) relative to those without RB1 mutations. For ocular clinical presentations, RB1-mutated retinoblastomas presented with a larger proportion of ectropion uveae (p = 0.017) and iris neovascularization (p = 0.001). These RB1 mutations comprised of 13 (24.1%) nonsense mutation, 13 (24.1%) splicing mutations, 11 (20.4%) frameshift deletions, 11 (20.4%) gross mutations, 3 (5.6%) missense mutations, 2 (3.7%) promoter mutations and 1 (1.9%) non-frameshift deletion. In addition, 8 novel RB1 mutations were identified. These germline RB1 mutations were not related to age at diagnosis or laterality. Here, we provide a comprehensive spectrum of RB1 germline mutations in Chinese Rb patients and describe correlations between RB1 mutations and clinical presentations. Our study also provides new evidence that will inform management and genetic counselling of Rb patients and families.

Turning copper metal into a Weyl semimetal

A search for new topological quantum systems is challenging due to the requirement of nontrivial band connectivity that leads to protected surface states of electrons. Progress in this field was primarily due to a realization of a band inversion mechanism between even and odd parity states that was proven to be very useful in both predicting many such systems and our understanding of their topological properties. Despite many proposed materials that assume the band inversion between $s$ and $p$ (or $p/d$, $d$/$f$) electrons, here, we explore a different mechanism where the occupied $d$ states subjected to a tetrahedral crystal field produce an active ${t}_{2g}$ manifold behaving as a state with an effective orbital momentum equal to $\ensuremath{-}1$, and pushing ${j}_{\mathrm{eff}}=1/2$ doublet at a higher energy. Via hybridization with nearest-neighbor orbitals realizable, e.g., in a zinc-blende structural environment, this allows a formation of odd parity state whose subsequent band inversion with an unoccupied $s$ band becomes possible, prompting us to look for the compounds with ${\mathrm{Cu}}^{+1}$ ionic state. Chemical valence arguments coupled to a search in the materials database of zinc-blende-like lattice space groups ${T}_{d}^{2}$ ($F\overline{4}3m$) lead us to systematically investigate electronic structures and topological properties of CuY ($Y=\text{F}$, Cl, Br, I) and $\mathrm{Cu}X\mathrm{O}$ ($X=\text{Li}$, Na, K, Rb) families of compounds. Our theoretical results show that CuF displays a behavior characteristic of an ideal Weyl semimetal with 24 Weyl nodes at the bulk Brillouin zone. We also find that other compounds, CuNaO and CuLiO, are the $s\text{\ensuremath{-}}d$ inversion-type topological insulators. Results for their electronic structures and corresponding surfaces states are presented and discussed in the context of their topological properties.

A Parent-of-Origin Effect Impacts the Phenotype in Low Penetrance Retinoblastoma Families Segregating the c.1981C>T/p.Arg661Trp Mutation of RB1.

Retinoblastoma (Rb), the most common pediatric intraocular neoplasm, results from inactivation of both alleles of the RB1 tumor suppressor gene. The second allele is most commonly lost, as demonstrated by loss of heterozygosity studies. RB1 germline carriers usually develop bilateral tumors, but some Rb families display low penetrance and variable expressivity. In order to decipher the underlying mechanisms, 23 unrelated low penetrance pedigrees segregating the common c.1981C>T/p.Arg661Trp mutation and other low penetrance mutations were studied. In families segregating the c.1981C>T mutation, we demonstrated, for the first time, a correlation between the gender of the transmitting carrier and penetrance, as evidenced by Fisher’s exact test: the probability of being unaffected is 90.3% and 32.5% when the mutation is inherited from the mother and the father, respectively (p-value = 7.10−7). Interestingly, a similar correlation was observed in families segregating other low penetrance alleles. Consequently, we investigated the putative involvement of an imprinted, modifier gene in low penetrance Rb. We first ruled out a MED4-driven mechanism by MED4 methylation and expression analyses. We then focused on the differentially methylated CpG85 island located in intron 2 of RB1 and showing parent-of-origin-specific DNA methylation. This differential methylation promotes expression of the maternal c.1981C>T allele. We propose that the maternally inherited c.1981C>T/p.Arg661Trp allele retains sufficient tumor suppressor activity to prevent retinoblastoma development. In contrast, when the mutation is paternally transmitted, the low residual activity would mimic a null mutation and subsequently lead to retinoblastoma. This implies that the c.1981C>T mutation is not deleterious per se but needs to be destabilized in order to reach pRb haploinsufficiency and initiate tumorigenesis. We suggest that this phenomenon might be a general mechanism to explain phenotypic differences in low penetrance Rb families.

Abstract B19: RB function as a central component of osteosarcoma behavior: A comparative assessment in dogs and humans

Abstract Introduction: There remain major gaps in our understanding of the pathogenesis of osteosarcoma (OS). This diagnosis represents a group of highly heterogeneous tumors, and an unresolved problem in their management is the lack of robust prognostic and predictive factors. Neither topography nor morphology, nor other conventional laboratory and ancillary tests can distinguish between highly aggressive and more indolent types of OS. Spontaneous OS is commonly diagnosed in dogs, and we showed recently that human and canine OS achieve similar phenotypes by altering convergent molecular pathways. Specifically canine and human OS can be stratified into two prognostically significant subgroups with inversely correlated expression of two gene clusters. This pattern was revealed only by the use of canine samples that also eliminated stromal signatures, as it was masked in studies that used human tumors or that employed different analyses. These two prognostic groups have significantly different behavior defined by survival, as well as by their ability to engraft and metastasize in mice. Equally important, there is exquisite overlap with a predictive signature called CINSARC (Complexity Index in SARComa) of soft tissue sarcomas, suggesting that OS can be used as proof of concept to support the canine model more generally for studies of sarcoma pathogenesis and therapy. Experimental Procedures: We used bioinformatics and genetic complementation, respectively, to identify and validate driver events that are responsible for the biological behavior of canine and human OS. The MEME Suite and Ingenuity Pathway Analysis were used for de novo motif discovery and to identify transcription factors with significant target enrichment in the prognostic signature. Immunoblotting, dual luciferase reporter assays, mutagenesis assays, and qRT-PCR were used to confirmed differential expression and activity of RB protein in cell lines derived from aggressive and indolent OS. Co-immunoprecipitation and ChIP were used to characterize complexes associated with endogenous or ectopic RB. Gene expression microarrays and bioinformatics were used to assess changes in genome wide gene expression after restoration of RB in aggressive OS. New Unpublished Data: The promoter sequences of all of the genes in the prognostic signature contained variants of RB-E2F-DP-1 binding sites. The gene signature also was significantly enriched for targets regulated by the RB and E2F families. RB expression was not the sole determinant of RB function, but genetic restoration of functional RB reversed the phenotype of each aggressive cell line analyzed to one resembling indolent tumors or normal osteoblasts, and it inevitably led to cell death. In contrast, indolent tumors that retained endogenous RB function were highly tolerant of RB addition, showing no (or minimal) phenotypic changes upon expression of ectopic RB. Somewhat surprisingly, restoration of RB also corrected other gene defects associated with the aggressive OS phenotype, such as activity of TP53 pathway. Conclusions: Taken together, our results suggest that RB inactivation is not necessary for the development of OS, but the integrity of the RB pathway is an important factor that contributes to behavior, clinical progression, and outcome for dogs and humans with OS. Our ongoing work seeks to develop pharmacological agents that mimic RB-dependent gene expression and chromatin organization that can be used to improve outcomes for patients with the most aggressive forms of OS. Citation Format: Milcah C. Scott, Aaron L. Sarver, Frances Phan, Rachit Gupta, Venugopal Thayanithy, Subbaya Subramanian, Jaime F. Modiano. RB function as a central component of osteosarcoma behavior: A comparative assessment in dogs and humans. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B19.

RB1 mutation spectrum in a comprehensive nationwide cohort of retinoblastoma patients

BackgroundRetinoblastoma (Rb) is a childhood cancer of the retina, commonly initiated by biallelic inactivation of the RB1 gene. Knowledge of the presence of a heritable RB1 mutation can help in...

Structural and Functional Analysis of Cyclin D1 Reveals p27 and Substrate Inhibitor Binding Requirements

An alternative strategy for inhibition of the cyclin dependent kinases (CDKs) in antitumor drug discovery is afforded through the substrate recruitment site on the cyclin positive regulatory subunit. Critical CDK substrates such as the Rb and E2F families must undergo cyclin groove binding before phosphorylation, and hence inhibitors of this interaction also block substrate specific kinase activity. This approach offers the potential to generate highly selective and cell cycle specific CDK inhibitors and to reduce the inhibition of transcription mediated through CDK7 and 9, commonly observed with ATP competitive compounds. While highly potent peptide and small molecule inhibitors of CDK2/cyclin A, E substrate recruitment have been reported, little information has been generated on the determinants of inhibitor binding to the cyclin groove of the CDK4/cyclin D1 complex. CDK4/cyclin D is a validated anticancer drug target and continues to be widely pursued in the development of new therapeutics based on cell cycle blockade. We have therefore investigated the structural basis for peptide binding to its cyclin groove and have examined the features contributing to potency and selectivity of inhibitors. Peptidic inhibitors of CDK4/cyclin D of pRb phosphorylation have been synthesized, and their complexes with CDK4/cyclin D1 crystal structures have been generated. Based on available structural information, comparisons of the cyclin grooves of cyclin A2 and D1 are presented and provide insights into the determinants for peptide binding and the basis for differential binding and inhibition. In addition, a complex structure has been generated in order to model the interactions of the CDKI, p27(KIP)¹, with cyclin D1. This information has been used to shed light onto the endogenous inhibition of CDK4 and also to identify unique aspects of cyclin D1 that can be exploited in the design of cyclin groove based CDK inhibitors. Peptidic and nonpeptidic compounds have been synthesized in order to explore structure-activity relationship for binding to the cyclin D1 groove, which to date has not been carried out in a systematic fashion. Collectively, the data presented provide new insights into how compounds can be developed that function as chemical biology probes to determine the cellular and antitumor effects of CDK inhibition. Furthermore, such compounds will serve as templates for structure-guided efforts to develop potential therapeutics based on selective inhibition of CDK4/cyclin D activity.

Tandem E2F Binding Sites in the Promoter of the p107 Cell Cycle Regulator Control p107 Expression and Its Cellular Functions

The retinoblastoma tumor suppressor (Rb) is a potent and ubiquitously expressed cell cycle regulator, but patients with a germline Rb mutation develop a very specific tumor spectrum. This surprising observation raises the possibility that mechanisms that compensate for loss of Rb function are present or activated in many cell types. In particular, p107, a protein related to Rb, has been shown to functionally overlap for loss of Rb in several cellular contexts. To investigate the mechanisms underlying this functional redundancy between Rb and p107 in vivo, we used gene targeting in embryonic stem cells to engineer point mutations in two consensus E2F binding sites in the endogenous p107 promoter. Analysis of normal and mutant cells by gene expression and chromatin immunoprecipitation assays showed that members of the Rb and E2F families directly bound these two sites. Furthermore, we found that these two E2F sites controlled both the repression of p107 in quiescent cells and also its activation in cycling cells, as well as in Rb mutant cells. Cell cycle assays further indicated that activation of p107 transcription during S phase through the two E2F binding sites was critical for controlled cell cycle progression, uncovering a specific role for p107 to slow proliferation in mammalian cells. Direct transcriptional repression of p107 by Rb and E2F family members provides a molecular mechanism for a critical negative feedback loop during cell cycle progression and tumorigenesis. These experiments also suggest novel therapeutic strategies to increase the p107 levels in tumor cells.

Regulation of RB Transcription In Vivo by RB Family Members

In cancer cells, the retinoblastoma tumor suppressor RB is directly inactivated by mutation in the RB gene or functionally inhibited by abnormal activation of cyclin-dependent kinase activity. While variations in RB levels may also provide an important means of controlling RB function in both normal and cancer cells, little is known about the mechanisms regulating RB transcription. Here we show that members of the RB and E2F families bind directly to the RB promoter. To investigate how the RB/E2F pathway may regulate Rb transcription, we generated reporter mice carrying an eGFP transgene inserted into a bacterial artificial chromosome containing most of the Rb gene. Expression of eGFP largely parallels that of Rb in transgenic embryos and adult mice. Using these reporter mice and mutant alleles for Rb, p107, and p130, we found that RB family members modulate Rb transcription in specific cell populations in vivo and in culture. Interestingly, while Rb is a target of the RB/E2F pathway in mouse and human cells, Rb expression does not strictly correlate with the cell cycle status of these cells. These experiments identify novel regulatory feedback mechanisms within the RB pathway in mammalian cells.

HP1α guides neuronal fate by timing E2F-targeted genes silencing during terminal differentiation

A critical step of neuronal terminal differentiation is the permanent withdrawal from the cell cycle that requires the silencing of genes that drive mitosis. Here, we describe that the alpha isoform of the heterochromatin protein 1 (HP1) protein family exerts such silencing on several E2F-targeted genes. Among the different isoforms, HP1alpha levels progressively increase throughout differentiation and take over HP1gamma binding on E2F sites in mature neurons. When overexpressed, only HP1alpha is able to ensure a timed repression of E2F genes. Specific inhibition of HP1alpha expression drives neuronal progenitors either towards death or cell cycle progression, yet preventing the expression of the neuronal marker microtubule-associated protein 2. Furthermore, we provide evidence that this mechanism occurs in cerebellar granule neurons in vivo, during the postnatal development of the cerebellum. Finally, our results suggest that E2F-targeted genes are packaged into higher-order chromatin structures in mature neurons relative to neuroblasts, likely reflecting a transition from a 'repressed' versus 'silenced' status of these genes. Together, these data present new epigenetic regulations orchestrated by HP1 isoforms, critical for permanent cell cycle exit during neuronal differentiation.

Expression of the E2F and retinoblastoma families of proteins during neural differentiation

Development of the brain is determined by a strictly orchestrated program of proliferation, migration, apoptosis, differentiation, synaptogenesis, tract formation, and myelination. The E2F family of transcription factors, whose activity and functions are regulated in large part through interactions with the retinoblastoma (Rb) family of tumor suppressor proteins, has been implicated as a key regulator of proliferation, differentiation, and apoptosis in a variety of tissues. We have examined levels of the E2F and Rb families of proteins during both brain development and neural differentiation of P19 cells, and found the expression profiles during these two processes of neural development and maturation to be quite similar, i.e., strong up-regulation of p130, pronounced down-regulation of p107, moderate up-regulation of pRb, and significant down-regulation of most species of E2F and dimerization protein (DP). However, several specific isoforms, namely a 30 kDa form of DP-2, a 57 kDa species of E2F-3, a 59 kDa form of E2F-5 and the isoforms of E2F-1 recognized by the E2F-1 (KH-95) antibody were up-regulated suggesting that these particular isoforms of E2F and DP play a tissue-specific function in differentiation and maturation of nervous tissue. The potential role of the E2F/DP family of transcription factors in aspects of neural development and differentiation are considered.

Accumulation of human papillomavirus type 16 E7 protein bypasses G1 arrest induced by serum deprivation and by the cell cycle inhibitor p21.

The E7 oncoproteins encoded by the high-risk type of human papillomaviruses (HPVs) interact with the Rb family proteins Rb, p107, and p130. The Rb family proteins associate with the factors of the E2F family to form transcription repressor complexes, which control expression of several genes essential for S-phase entry and DNA replication. The E7 oncoproteins, by interacting with the Rb family proteins, dissociate the repressor complexes involving the factors of the E2F and Rb families, leading to a release of the E2F factors in their activator forms. In this study, we have addressed the mechanism by which the HPV type 16 (HPV16) E7 stimulates the cell cycle. Using a cell line that inducibly expresses the HPV16 E7 protein, we show that an accumulation of E7 induces quiescent cells to enter S phase and that this function of E7 depends on retention of the motif involved in binding to the Rb family proteins. To study the effects of E7 on normal human cells, we generated a recombinant adenovirus that expresses the HPV16 E7 protein. Infection of normal human fibroblasts, which were arrested in G1 phase by serum deprivation, with the E7-expressing virus induced the cells to enter S phase. The E7-induced S phase entry was accompanied by an increase in the activator form of E2F, but no increase in the cyclin-dependent kinase (cdk) activity was detected. Infection of serum-stimulated fibroblasts with a recombinant adenovirus expressing the cdk inhibitor p21 inhibited progression into S phase. Coinfection with the E7-expressing virus abrogated the p21 inhibition of progression into S phase without increasing the cdk activity. These results are consistent with the notion that E7 stimulates entry into S phase through targets downstream of the cdks such as the proteins of the E2F and Rb families.

Regulation and Expression of Retinoblastoma Proteins p107 and p130 during 3T3-L1 Adipocyte Differentiation

During 3T3-L1 adipocyte differentiation, growth-arrested, postconfluent preadipocytes are required to reenter the cell cycle and proceed through a mitotic clonal expansion phase prior to terminal differentiation. The retinoblastoma proteins (pRB, p107, and p130) are thought to be critical in controlling cell cycle progression by binding to and regulating the activity of the E2F transcription factors. We show here that p130/p107 protein levels, p107 mRNA levels, and E2F DNA binding complexes are regulated during 3T3-L1 adipogenesis. The predominant E2F binding complex in day 0 preadipocytes was p130-E2F with no detectable free E2F or p107. On Day 1, during mitotic clonal expansion, there was a distinct switch to free E2F and p107-E2F complexes associated with increased p107 mRNA and protein along with decreased p130 protein levels. Following differentiation, the day 0 pattern is reestablished. The switch is not just a consequence of reentry into the cell cycle, in that p107 protein levels are both detectable and unchanged in dividing, serum-restricted, or serum restimulated preconfluent cells. Interestingly, hormonal stimulation of 3T3-C2 cells, a related nondifferentiating cell line, also induces a mitotic clonal expansion phase that is associated with the p130:p107 switch in a pattern very similar to 3T3-L1 cells, suggesting the block in differentiation observed in 3T3-C2 cells occurs after clonal expansion. Combined, these findings suggest that the regulatory mechanisms of the p130:p107 switch are not specific to differentiation but may play a key role in regulating the mitotic clonal expansion necessary for adipocyte differentiation in 3T3-L1 cells.

Applications cliniques du diagnostic moléculaire du rétinoblastome dans 15 families

In 40% retinoblastoma (Rb) results from a hereditary mutation of the Rb susceptibility gene (RB1). In this study, we tested the usefulness of intragenic DNA analysis for ophthalmologic follow-up in affected families. Molecular analysis was performed on 103 DNA samples of 15 Rb families. We used 7 intragenic polymorphic markers and one within the ESD gene for mutation linkage analysis. DNA analysis was informative in 88% of relatives at risk of developing Rb. Among them, the presence of a mutated RB1 allele was excluded in 46%, while 29% were unaffected carriers and 25% had inherited the Rb predisposition. In the majority of familial Rb, the DNA analysis allows the identification of children carrying a RB1 mutation and who will need a close ophthalmologic follow-up under general anesthesia. When the mutated gene is absent, ophthalmological examination under narcosis is unnecessary. Finally, identification of asymptomatic carriers improve the accuracy of genetic counselling.