RB1 Alleles Research Articles

Abstract A24: Brain tumor induction after somatic inactivation of rb1 in zebrafish

Abstract Purpose: We found that targeted somatic inactivation of rb1 leads to solid tumor formation in the central nervous system of adult zebrafish. The purpose of our study was to characterize the histological and genetic features of the tumors. Methods: Zebrafish rb1 tumor suppressor was inactivated in somatic tissue by injection of transcription activator-like effector nucleases (TALENs) targeting exon 2 and exon 3 at the one-cell stage. Histopathology and immunohistochemistry with s100 and pH3 was performed on tumor tissue. To confirm TALEN-mediated editing of the targeted rb1 loci and characterize the frequency and type of indel alleles in tumor and somatic tissues we sequenced cloned amplicons from each locus. Results: Zebrafish injected with TALENs targeting exon 2 or exon 3 of rb1 presented with cranial tumors beginning at six months of age. By one year the frequency of adults with tumors was 33-50% and 11%, respectively. Histopathological analyses of tumors from eight exon 2-targeted and six exon 3-targeted fish revealed a range of undifferentiated and differentiated tumor phenotypes. Seven tumors exhibited features suggestive of primitive neuroectodermal tumors (PNET). The remainder exhibited more differentiated tumor phenotypes resembling glioma. S100 was detected in a subset of the tumors. pH3 labeled regions of high mitotic activity in each of the tumors. In 4 out of 4 tumors the frequency of rb1 mutant alleles (61%-87% of cloned amplicons) was consistently higher than wild type. However, only one or two specific rb1 alleles were present in 3 out of 4 tumors. In contrast, the mutation frequency in retinal, muscle, and germline tissues was highly variable (3%-100%), and represented by up to seven different mutant rb1 alleles. Conclusion: Somatic inactivation of rb1 resulted in brain tumors showing varying degrees of differentiation suggesting the possibility that transformed neural progenitors give rise to tumors. rb1 allele frequency is consistent with clonal expansion contributing to tumor growth. Predisposing zebrafish to brain tumors by somatic inactivation of rb1 with site-specific nucleases suggests a simple method for investigating pathways cooperating in tumor progression. Citation Format: Staci L. Solin, Heather R. Shive, Jeffrey J. Essner, Maura McGrail. Brain tumor induction after somatic inactivation of rb1 in zebrafish. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A24.

Receptor tyrosine kinase Kit action in skin melanocytes: is it exclusively cell autonomous?

Receptor tyrosine kinase Kit action in skin melanocytes: is it exclusively cell autonomous?

Abstract 2766: Advanced paternal age as a possible risk factor for retinoblastoma

Abstract Retinoblastoma is a pediatric tumor arising in the retina with an incidence of 1 in 15000 to 18000 live births. Retinoblastoma is initiated by the inactivation of both the alleles of RB1 and it may be inherited or sporadic. There are several possible risk factors suggested for sporadic retinoblastoma including advanced paternal age.We evaluated the father's age in 42 children with retinoblastoma attending the Aravind Eye Hospital, Madurai, India from Mar 2013 to Feb 2014 retrospectively. Fathers with age 40 and above at the time of conception were included in the analysis. Patients with a positive family history were excluded from the analysis. Genetic analysis of RB1 was carried out in the tumor and blood samples of these patients of older father and their family.There were 5 patients (3 unilateral and 2 bilateral) having fathers with age 40 years and above. Genetic analysis identified RB1 mutations in the tumor samples of 4 patients and these mutations were not present in the blood samples of patient and family members. Since all these patients had somatic mutations, the involvement of paternal age impact was ruled out. In blood sample of one bilateral patient, we found de novo germline RB1 mutation possibly suggesting a role of advanced paternal age.In conclusion, the advanced paternal age seems to be a minor risk factor for retinoblastoma. Further study of large number of patients may provide a better understanding of the impact of advanced paternal age in retinoblastoma. Citation Format: Vanniarajan Ayyasamy, Namrata Gaikwad, Thirumalairaj Kannan, Aloysius A. Abraham, Veerappan Muthukkaruppan, Usha Kim. Advanced paternal age as a possible risk factor for retinoblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2766. doi:10.1158/1538-7445.AM2015-2766

The Biology of Retinoblastoma.

Retinoblastoma, the most common primary intraocular cancer of childhood, is a malignancy arising in the developing retina. Tumor formation usually begins with mutation in both alleles of the retinoblastoma tumor suppressor gene RB1, followed by a series of other genetic alterations that correlate with the clinical stage and pathologic findings of the tumor. Analysis of sporadic and heritable retinoblastoma led to the development of Knudson's Two-Hit Hypothesis. The tumor suppressor RB1 gene codes for the retinoblastoma protein which is a key regulator of cellular replication via its binding to the E2F family of transcription factors and chromatin remodeling proteins. Studies of preclinical models of retinoblastoma in the form of transgenic mice and xenograft animal models have significantly contributed to the development of effective therapies for this disease. Research on retinoblastoma has paved the way toward understanding many of the mechanisms in cancer genetics.

Abstract 3623: Fluctuating antibody response and CD4-positive T-cells in a small-cell lung cancer mouse model

Abstract Patients with small-cell lung cancer (SCLC), a highly metastatic type of lung cancer, show a dismal 5-year survival of only 5%. Effective therapies are urgently needed. Notably, about fifteen-percent of SCLC-patients harbor anti-Hu autoantibodies, which are associated with improved survival. This suggests that the anti-Hu response might be harnessed for immunotherapy. A conditional SCLC mouse model with floxed Rb1 and p53 alleles, in which the cancer is induced by instilling Cre-recombinase into the lungs via intratracheal intubation, offers a unique opportunity to study the immune response in detail. We have previously shown that, just like human SCLC patients, a fraction of these SCLC-carrying mice show an anti-Hu autoantibody response. Here we examine the presence of anti-Hu reactive T-cells using a prospective cohort (n=45) of SCLC-induced mice. All mice develop SCLC, showing systemic disease in 4-7 months following inactivation of floxed Rb1 and p53 alleles. Monthly autoantibody analysis by western blotting revealed an antibody response in a subset of mice, sometimes as early as 1 month after induction. Reactivity usually peaked approximately 3-4 months after induction and declined as disease progressed. This suggests an immune escape mechanism and/or antibody adsorption by the rapidly proliferating tumors. T-cells were isolated at euthanasia from the spleens of moribund mice. Proliferating anti-HuD CD4-positive T-cells were detected in fifty-one-percent of mice (eighteen out of thirty-five) by flow cytometry. Anti-HuD T-cell proliferation from spleen cells was validated in a subsequent prospective cohort (n=45) of SCLC-induced mice using thymidine incorporation. Stratifying for antibody positive and negative mice using an stringent cutoff, there was no significant difference in T cell response between the two groups (p=0.11), however four mice in the antibody positive group showed the most elevated anti-HuD T-cell response. Our observations warrant a much larger study, in which in-depth characterization of the humoral and cellular the anti-HuD immune response is carried out at multiple time points and correlated with a detailed analysis of the stage of tumor development and mestastasis. Preliminary data hints at a negative correlation between the anti-HuD antibody and T-cell response on one hand and tumor size and metastasis on the other. The observation that the Hu antigen in mice with SCLC is a target for both humoral and cellular immune responses underscores the utility of this mouse model in the preclinical evaluation the anti-Hu immune response for therapy and clinical translation. Citation Format: Mario A. Pulido, Vincent Lombardi, Diane Lee, Yiwei Wang, Eric Chung, Lina Wang, W. Martin Kast, Omid Akbari, Ite A. Laird-Offringa. Fluctuating antibody response and CD4-positive T-cells in a small-cell lung cancer mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3623. doi:10.1158/1538-7445.AM2014-3623

Abstract 4402: Synergistic cell death in nedd8 and hrb27c mutant cells under an rbf-negative background is dependent on EGFR/MAPK signaling

Abstract The Rb (retinoblastoma) protein is highly functionally conserved between the Drosophila, and deregulation and mutations in the Rb pathway are common events across a wide range of cancers. Here, we identify two novel synthetic lethal interactions between Rb and mutant alleles of Nedd8 and Hrb27c that were identified through an unbiased genetic screen in Drosophila that resulted in apoptosis in both the wing disc pouch and the region of the eye disc posterior to the morphogenetic furrow. However, the molecular mechanisms behind the apoptotic phenotypes in both synthetic lethal double mutants have yet to be elucidated. It is known that wild-type Rb normally sequesters and can prevent the E2F1 transcription factor from promoting transcription of hid, the major pro-apoptotic regulator in imaginal discs. Synthetic lethality is therefore likely to be contingent on EGFR/hid signaling, and I will determine if such is the case through a series of genetic epistasis tests. Interestingly, preliminary evidence also suggests that EGFR/MAPK signaling may be ablated in both mutants of Nedd8 and Hrb27c. However, this observation is completely novel and therefore the molecular mechanisms behind downregulation of MAPK signaling have yet to be elucidated in both these mutants. I will again perform a series of epistasis experiments to determine how Nedd8 and Hrb27c mutations mechanistically affect EGFR/MAPK signaling. This will be achieved first by determining if signaling is downregulated either during EGFR ligand processing or downstream of the EGFR receptor. This will be followed by subsequent experiments to determine which specific element in either pathway is being affected by Nedd8 or Hrb27c ablation. I therefore hypothesize that inhibition of both nedd8 and hrb27c synergistically induce cell death in Rb-negative cells in a process that is dependent on both E2F1 activity and downregulation of MAPK signaling. Many existing treatment regimens fail to take into account the unique biological properties of cancer cells compared to their normal counterparts, thus often resulting in off-target effects and limitations in dosage. This proposal is therefore both novel and significant because the mechanisms behind both the Rb/Hrb27c and Rb/Nedd8 synthetic lethal interactions have yet to be elucidated and characterization of such interactions such as these have the potential to catalyze the development of new therapeutic strategies in the treatment of cancer. Note: This abstract was not presented at the meeting. Citation Format: Robin Zhang, Tianyi Zhang, Zhentao Sheng, Wei Du. Synergistic cell death in nedd8 and hrb27c mutant cells under an rbf-negative background is dependent on EGFR/MAPK signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4402. doi:10.1158/1538-7445.AM2014-4402

Characterization of the genomic architecture and mutational spectrum of a small cell prostate carcinoma.

We present the use of a series of laboratory, analytical and interpretation methods to investigate personalized cancer care for a case of small cell prostate carcinoma (SCPC), a rare and aggressive tumor with poor prognosis, for which the underlying genomic architecture and mutational spectrum has not been well characterized. We performed both SNP genotyping and exome sequencing of a Virchow node metastasis from a patient with SCPC. A variety of methods were used to analyze and interpret the tumor genome for copy number variation, loss of heterozygosity (LOH), somatic mosaicism and mutations in genes from known cancer pathways. The combination of genotyping and exome sequencing approaches provided more information than either technique alone. The results showed widespread evidence of copy number changes involving most chromosomes including the possible loss of both alleles of CDKN1B (p27/Kip1). LOH was observed for the regions encompassing the tumor suppressors TP53, RB1, and CHD1. Predicted damaging somatic mutations were observed in the retained TP53 and RB1 alleles. Mutations in other genes that may be functionally relevant were noted, especially the recently reported high confidence cancer drivers FOXA1 and CCAR1. The disruption of multiple cancer drivers underscores why SCPC may be such a difficult cancer to manage.

A Rb1 promoter variant with reduced activity contributes to osteosarcoma susceptibility in irradiated mice.

BackgroundSyndromic forms of osteosarcoma (OS) account for less than 10% of all recorded cases of this malignancy. An individual OS predisposition is also possible by the inheritance of low penetrance alleles of tumor susceptibility genes, usually without evidence of a syndromic condition. Genetic variants involved in such a non-syndromic form of tumor predisposition are difficult to identify, given the low incidence of osteosarcoma cases and the genetic heterogeneity of patients. We recently mapped a major OS susceptibility QTL to mouse chromosome 14 by comparing alpha-radiation induced osteosarcoma in mouse strains which differ in their tumor susceptibility.MethodsTumor-specific allelic losses in murine osteosacoma were mapped along chromosome 14 using microsatellite markers and SNP allelotyping. Candidate gene search in the mapped interval was refined using PosMed data mining and mRNA expression analysis in normal osteoblasts. A strain-specific promoter variant in Rb1 was tested for its influence on mRNA expression using reporter assay.ResultsA common Rb1 allele derived from the BALB/cHeNhg strain was identified as the major determinant of radiation-induced OS risk at this locus. Increased OS-risk is linked with a hexanucleotide deletion in the promoter region which is predicted to change WT1 and SP1 transcription factor-binding sites. Both in-vitro reporter and in-vivo expression assays confirmed an approx. 1.5 fold reduced gene expression by this promoter variant. Concordantly, the 50% reduction in Rb1 expression in mice bearing a conditional hemizygous Rb1 deletion causes a significant rise of OS incidence following alpha-irradiation.ConclusionThis is the first experimental demonstration of a functional and genetic link between reduced Rb1 expression from a common promoter variant and increased tumor risk after radiation exposure. We propose that a reduced Rb1 expression by common variants in regulatory regions can modify the risk for a malignant transformation of bone cells after radiation exposure.

Conditional deletion of p53 and Rb in the renin-expressing compartment of the pancreas leads to a highly penetrant metastatic pancreatic neuroendocrine carcinoma

Efforts to model human pancreatic neuroendocrine tumors (PanNETs) in animals have been moderately successful, with minimal evidence for glucagonomas or metastatic spread. The renin gene, although classically associated with expression in the kidney, is also expressed in many other extrarenal tissues including the pancreas. To induce tumorigenesis within rennin-specific tissues, floxed alleles of p53 and Rb were selectively abrogated using Cre-recombinase driven by the renin promoter. The primary neoplasm generated is a highly metastatic islet cell carcinoma of the pancreas. Lineage tracing identifies descendants of renin-expressing cells as pancreatic alpha cells despite a lack of active renin expression in the mature pancreas. Both primary and metastatic tumors express high levels of glucagon; furthermore, an increased level of glucagon is found in the serum, identifying the pancreatic cancer as a functional glucagonoma. This new model is highly penetrant and exhibits robust frequency of metastases to the lymph nodes and the liver, mimicking human disease, and provides a useful platform for better understanding pancreatic endocrine differentiation and development, as well as islet cell carcinogenesis. The use of fluorescent reporters for lineage tracing of the cells contributing to disease initiation and progression provides an unique opportunity to dissect the timeline of disease, examining mechanisms of the metastatic process, as well as recovering primary and metastatic cells for identifying cooperating mutations that are necessary for progression of disease.

Retinoblastoma protein prevents enteric nervous system defects and intestinal pseudo-obstruction.

The retinoblastoma 1 (RB1) tumor suppressor is a critical regulator of cell cycle progression and development. To investigate the role of RB1 in neural crest-derived melanocytes, we bred mice with a floxed Rb1 allele with mice expressing Cre from the tyrosinase (Tyr) promoter. TyrCre+;Rb1fl/fl mice exhibited no melanocyte defects but died unexpectedly early with intestinal obstruction, striking defects in the enteric nervous system (ENS), and abnormal intestinal motility. Cre-induced DNA recombination occurred in all enteric glia and most small bowel myenteric neurons, yet phenotypic effects of Rb1 loss were cell-type specific. Enteric glia were twice as abundant in mutant mice compared with those in control animals, while myenteric neuron number was normal. Most myenteric neurons also appeared normal in size, but NO-producing myenteric neurons developed very large nuclei as a result of DNA replication without cell division (i.e., endoreplication). Parallel studies in vitro found that exogenous NO and Rb1 shRNA increased ENS precursor DNA replication and nuclear size. The large, irregularly shaped nuclei in NO-producing neurons were remarkably similar to those in progeria, an early-onset aging disorder that has been linked to RB1 dysfunction. These findings reveal a role for RB1 in the ENS.

Abstract 3863: Transgenic model for early prostate metastasis to the lymph nodes.

Abstract Background: The emergence of recurrent, metastatic prostate cancer following the failure of androgen-deprivation therapy represents the lethal phenotype of this disease. However, little is known regarding the genes and pathways that regulate this metastatic process, and moreover, it is unclear whether metastasis is an early or late event. The SSeCKS/Gravin/AKAP12 (“SSeCKS”) gene, previously shown to suppress prostate cancer metastasis upon re-expression (1,2), as well as the tumor suppressor gene, Rb, are either transcriptionally downregulated or deleted in human prostate cancers (3,4). Mouse models deficient in either SSeCKS or Rb exhibit prostatic hyperplasia (5,6). Moreover, SSeCKS-null fibroblasts as well as SSeCKS-null prostates display Rb-dependent premature senescence markers (7). Methods: In order to determine whether the combined loss of AKAP12 and Rb in the prostate synergizes to induce oncogenic progression, Akap12-/- mice were crossed with Pb4-Cre;RbloxP/loxP mice to generate Akap12-/-;RbPE-/− progeny, with the Probasin-Cre passed only through males. Results: The combined loss of SSeCKS and Rb results in prostatic intraepithelial neoplasia (PIN) starting at 6 months of age that fails to progress to malignancy (adenocarcinoma) after 18 months. The PIN lesions were marked by increased Ki-67 proliferation of cytokeratin 8 (CK8), p63-negative luminal cells as well as p63-positive basal cells. Interestingly, these lesions also had increased numbers of androgen-receptor (AR)-positive, p63-positive, CK5-negative cells. There was evidence of reactive stroma including mural hyperplasia and inflammatory cell infiltration. Strikingly, 83% of mice with PIN lesions exhibited metastases to draining lymph nodes (LN), marked by well-differentiated tumors cells expressing markers of basal (p63, CK14) and luminal (CK8 and AR) epithelial cells, although none expressed the basal marker, CK5. PCR-based tests for the deleted floxed Rb allele from laser capture microdissected LN lesions confirmed the prostatic origin of these metastatic cells. The LN lesions showed very limited inflammation, based on very few cells staining with the histiocyte marker, CD68. Conclusions: Taken together, these data suggest that in the context of Rb loss, SSeCKS suppresses the oncogenic proliferation and early metastatic spread of transitional, basal-luminal prostate tumor cells. Citation Format: Hyun-Kyung Ko, Shin Akakura, Jennifer Peresie, David W. Goodrich, Barbara A. Foster, Irwin H. Gelman. Transgenic model for early prostate metastasis to the lymph nodes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3863. doi:10.1158/1538-7445.AM2013-3863

Allelic loss of selected tumor suppressor genes in acute lymphoblastic leukemia in children

Defect in function of tumor suppressor genes may lead to initiation/progression of leukemias. RB1, CDKN2A and TP53 gene alterations are found in acute lymphoblastic leukemia (ALL) in children. Data showing a contribution of these alterations to the pathomechanism of leukemias are contradictory and their impact on a disease course still remains undefined. The main aim of the study was to identify and the characterize of RB1, CDKN2A and TP53 allele loss in ALL children patients at diagnosis. 46 children with de novo ALL were examined. Fluorescent in situ hybridization was performed on bone marrow smear preparations. We demonstrated that at least one of three investigated deletions occurred statistically more frequently in T-lineage leukemia patients (p = 0.044); this was the most frequent in respect to RB1 gene (p = 0.054). Additionally, at least one of the examined deletions was observed statistically more frequently in patients with WBC above 20 000/µl (p = 0.043), this was the most frequent for CDKN2A gene (p = 0.066). Presented results seem to give an evidence that deletions of RB1 and CDKN2A genes may contribute to the development of hyperleukocytic type of T-lineage ALL in children, nevertheless this observation needs further investigations.

Identificación de tres nuevas mutaciones en el gen RB1 en pacientes con retinoblastoma esporádico en Colombia

Retinoblastoma is a childhood cancer of the retina originated by altered or null retinoblastoma protein (pRb) expression. Genetic alterations in both RB1 alleles in the retinal cells are required for the development of retinoblastoma. In the sporadic form, non-hereditary RB1 gene mutations take place in a single retinoblast cell, and are therefore only present in tumor DNA (somatic mutations). Sporadic retinoblastoma is primarily unilateral, lacks family history and has no risk of transmission to descendants. Genetic tests for detection of RB1 mutation has improved the identification of carriers and facilitated accurate genetic counseling.To identify mutations in the RB1 gene in Colombian patients with sporadic retinoblastoma by PCR-SSCP followed by sequence.Four patients with sporadic retinoblastoma were analyzed by PCR-SSCP, followed by DNA sequencing to identify variations in the RB1 gene.We identified five variations in RB1 gene: three new mutations (one germline and two somatic mutations), one new polymorphism and one already reported somatic mutation. Four mutations were found in three patients with unilateral retinoblastoma and one mutation was found in a patient with bilateral retinoblastoma. One of these was a germline mutation in a sporadic unilateral retinoblastoma that was not present in the parents or three siblings analyzed.Our results emphasize the importance of identifying mutations for genetic counseling and clinical management of sporadic retinoblastoma patients. Description of a new RB1 gene variant is interesting since there have been a small number of polymorphisms reported for this gene.

Identification of three new mutations in the RB1 gene in patients with sporadic retinoblastoma in Colombia.

Retinoblastoma is a childhood cancer of the retina originated by altered or null retinoblastoma protein (pRb) expression. Genetic alterations in both RB1 alleles in the retinal cells are required for the development of retinoblastoma. In the sporadic form, non-hereditary RB1 gene mutations take place in a single retinoblast cell, and are therefore only present in tumor DNA (somatic mutations). Sporadic retinoblastoma is primarily unilateral, lacks family history and has no risk of transmission to descendants. Genetic tests for detection of RB1 mutation has improved the identification of carriers and facilitated accurate genetic counseling. To identify mutations in the RB1 gene in Colombian patients with sporadic retinoblastoma by PCR-SSCP followed by sequence. Four patients with sporadic retinoblastoma were analyzed by PCR-SSCP, followed by DNA sequencing to identify variations in the RB1 gene. We identified five variations in RB1 gene: three new mutations (one germline and two somatic mutations), one new polymorphism and one already reported somatic mutation. Four mutations were found in three patients with unilateral retinoblastoma and one mutation was found in a patient with bilateral retinoblastoma. One of these was a germline mutation in a sporadic unilateral retinoblastoma that was not present in the parents or three siblings analyzed. Our results emphasize the importance of identifying mutations for genetic counseling and clinical management of sporadic retinoblastoma patients. Description of a new RB1 gene variant is interesting since there have been a small number of polymorphisms reported for this gene.

Genetics of Primary Intraocular Tumors

Primary intraocular neoplasms are tumors that originate within the eye. The most common malignant primary intraocular tumor in adults is uveal melanoma and the second is primary intraocular lymphoma or vitreoretinal (intraocular) lymphoma. The most common malignant intraocular tumor in children is retinoblastoma. Genetics plays a vital role in the diagnosis and detection of ocular tumors. In uveal melanoma, monosomy 3 is the most common genetic alteration and somatic mutations of BAP1, a tumor suppressor gene, have been reported in nearly 50% of primary uveal melanomas. The retinoblastoma gene RB1 is the prototype tumor suppressor gene—mutations in RB1 alleles lead to inactivated RB protein and the development of retinoblastoma. Immunoglobulin heavy chain (IgH) or T-cell receptor (TCR) gene rearrangement is observed in B-cell or T-cell primary vitreoretinal lymphoma, respectively. Other factors related to the genetics of these three common malignancies in the eye are discussed and reviewed.

Fuzzy Tandem Repeats Containing p53 Response Elements May Define Species-Specific p53 Target Genes

Evolutionary forces that shape regulatory networks remain poorly understood. In mammals, the Rb pathway is a classic example of species-specific gene regulation, as a germline mutation in one Rb allele promotes retinoblastoma in humans, but not in mice. Here we show that p53 transactivates the Retinoblastoma-like 2 (Rbl2) gene to produce p130 in murine, but not human, cells. We found intronic fuzzy tandem repeats containing perfect p53 response elements to be important for this regulation. We next identified two other murine genes regulated by p53 via fuzzy tandem repeats: Ncoa1 and Klhl26. The repeats are poorly conserved in evolution, and the p53-dependent regulation of the murine genes is lost in humans. Our results indicate a role for the rapid evolution of tandem repeats in shaping differences in p53 regulatory networks between mammalian species.

Retinoblastoma Tumorigenesis: Genetic and Epigenetic Changes Walk Hand in Hand

The rate-limiting step in retinoblastoma tumorigenesis is inactivation of both RB1 alleles, but it has remained unclear how this tumor acquires the additional changes that constitute a malignant phenotype. Zhang et al. characterized the genetic and epigenetic alterations in four retinoblastomas using whole-genome analysis techniques. In these samples, the retinoblastoma genome was found to be remarkably stable genetically, although recurrent mutations in BCOR were identified in 13% of patients. However, an approach that integrated the results of ChIP, methylation and expression analysis identified multiple, more frequent alterations of the epigenetic landscape. One of the leading genes on the list the authors obtained was SYK, a kinase epigenetically upregulated. Knockdown of this gene and exposure to small molecules inhibiting the kinase function stopped tumor growth in vitro and in vivo, thus offering a new therapeutic target for the treatment of retinoblastoma.

More epigenetic hits than meets the eye: microRNAs and genes associated with the tumorigenesis of retinoblastoma

Retinoblastoma (RB), a childhood neoplasia of the retinoblasts, can occur unilaterally or bilaterally, with one or multiple foci per eye. RB is associated with somatic loss of function of both alleles of the tumor suppressor gene RB1. Hereditary forms emerge due to germline loss of function mutations in RB1 alleles. RB has long been the prototypic “model” cancer ever since Knudson's “two-hit” hypothesis. However, a simple two-hit model for RB is challenged by an increasing number of studies documenting additional hits that contribute to RB development. Here we review the genetics and epigenetics of RB with a focus on the role of small non-coding RNAs (microRNAs) and on novel findings indicating the relevance of DNA methylation in the development and prognosis of this neoplasia. Studies point to an elaborated landscape of genetic and epigenetic complexity, in which a number of events and pahtways play crucial roles in the origin and prognosis of RB. These include roles for microRNAs, inprinted loci, and parent-of-origin contributions to RB1 regulation and RB progression. This complexity is also manifested in the structure of the RB1 locus itself: it includes numerous repetitive DNA segments and retrotransposon insertion elements, some of which are actively transcribed from the RB1 locus. Altogether, we conclude that RB1 loss of function represents the tip of an iceberg of events that determine RB development, progression, severity, and disease risk. Comprehensive assessment of personalized RB risk will require genetic and epigenetic evaluations beyond RB1 protein coding sequences.

Influence of MDM2 and MDM4 on development and survival in hereditary retinoblastoma

Retinoblastoma (RB) accounts for 3% of all childhood malignancies, with different incidences around the world. This malignancy results from loss-of-function of both RB1 alleles although other genes, like MDM2 and MDM4, have been proposed to be involved in tumor development. We genotyped rs2279744T>G and rs937283A>G in MDM2, and rs4252668T>C and rs116197192G>A in MDM4, in 104 unrelated RB patients and 104 controls. Sixty-month survival Kaplan-Meier curves and χ(2)-tests were performed for estimating the putative effect of MDM2 and MDM4 alleles on disease progression and survival of RB patients. MDM2 rs2279744G was significantly more frequent in controls, indicating an apparently protective effect on RB development. However, survival of patients who carried a constitutional RB1 mutation was significantly lower with rs2279744TG or GG than with rs2279744TT. Presence of rs2279744G and a constitutional RB1 mutation was sixfold more frequent in the 0-12 month age group than other age groups at onset of symptoms (P = 0.0401). MDM4 rs4252668C was present at a significantly higher frequency in controls while the frequency of MDM4 rs116197192G was significantly higher in RB patients, suggesting that this allele might increase the risk of developing RB. Our results indicate that MDM2 and MDM4 polymorphisms may influence development and/or survival in RB.

Regulation of Retinoblastoma Protein (Rb) by p21 Is Critical for Adaptation to Massive Small Bowel Resection

BackgroundAdaptation following massive intestinal loss is characterized by increased villus height and crypt depth. Previously, we demonstrated that p21-null mice do not adapt after small bowel resection (SBR). As retinoblastoma protein (Rb) levels are elevated in p21-null crypt cells, we first sought to determine whether Rb is required for normal adaptation. Next, we tested whether Rb expression is responsible for blocked adaptation in p21-nulls. MethodsGenetically manipulated mice and wild-type (WT) littermates underwent either 50% SBR or sham operation. The intestine was harvested at 3, 7, or 28 days later and intestinal adaptation was evaluated. Enterocytes were isolated and protein levels evaluated by Western blot and quantified by optical density. ResultsRb-null mice demonstrated increased villus height, crypt depth, and proliferative rate at baseline, but there was no further increase following SBR. Deletion of one Rb allele lowered Rb expression and restored resection-induced adaptation responses in p21-null mice. ConclusionRb is specifically required for resection-induced adaptation. Restoration of adaptation in p21-null mice by lowering Rb expression suggests a crucial mechanistic role for Rb in the regulation of intestinal adaptation by p21.