The RB gene family controls the maturation state of the EndoC-ßH2 human pancreatic ß-cells.

The functional maturation of human pancreatic ß-cells remains poorly understood. EndoC-ßH2 is a human ß-cell line with a reversible immortalized phenotype. Removal of the two oncogenes, SV40LT and hTERT introduced for its propagation, stops proliferation, triggers cell size increase and senescence, promotes mitochondrial activity and amplifies several ß-cell traits and functions. Overall, these events recapitulate several aspects of functional ß-cell maturation. We report here that selective depletion of SV40LT, but not of hTERT, is sufficient to revert EndoC-ßH2 immortalization. SV40LT inhibits the activity of the RB family members and of P53. In EndoC-ßH2 cells, the knock-down of RB itself, and, to a lesser extent, of its relative P130, precludes most events triggered by SV40LT depletion. In contrast, the knock-down of P53 does not prevent reversion of immortalization. Thus, an increase in RB and P130 activity, but not in P53 activity, is required for functional maturation of EndoC-ßH2 cells upon SV40LT-depletion. In addition, RB and/or P130 depletion in SV40LT-expressing EndoC-ßH2 cells decreases cell size, stimulates proliferation, and decreases the expression of key ß-cell genes. Thus, despite SV40LT expression, EndoC-ßH2 cells have a residual RB activity, which when suppressed reverts them to a more immature phenotype. These results show that the expression and activity levels of RB family members, especially RB itself, regulate the maturation state of EndoC-ßH2 cells.

MiR-17-5p enhances pancreatic cancer proliferation by altering cell cycle profiles via disruption of RBL2/E2F4-repressing complexes.

The members of the miR-17-92 cluster are upregulated in various cancers and function as a cluster of oncogenic miRNA. Our study characterized a new function of miR-17-5p, a member of the miR-17-92 cluster, in regulating cell proliferation in pancreatic cancer. Our results indicate that miR-17-5p was up-regulated in pancreatic adenocarcinoma and directly targeted the retinoblastoma-like protein 2 (RBL2), a tumor suppressor belonging to the Rb family. High levels of miR-17-5p and low levels of RBL2 were associated with poor prognosis. RBL2 interacted with the transcription factor E2F4 and bound to the promoter regions of the E2F target genes. Disruption of the RBL2/E2F4 complex by miR-17-5p overexpression shifted the activity of E2F from gene repressing to gene activating, which induced cell cycle entry and proliferation. These results suggest that miR-17-5p promoted proliferation in pancreatic ductal adenocarcinoma cells (PDAC), and altered cell cycle profiles in vivo and in vitro, by disrupting the RBL2/E2F4-associated gene repressing complexes via direct targeting of RBL2. The new regulatory network, involving miR-17-5p and RBL2, emerges as a new target of PDAC treatment.

Genomic landscape of retinoblastoma in Rb-/- p130-/- mice resembles human retinoblastoma.

Several murine retinoblastoma models have been generated by deleting the genes encoding for retinoblastoma susceptibility protein pRb and one of its family members p107 or p130. In Rb-/- p107-/- retinoblastomas, somatic copy number alterations (SCNAs) like Mdm2 amplification or Cdkn2a deletion targeting the p53-pathway occur, which is uncommon for human retinoblastoma. In our study, we determined SCNAs in retinoblastomas developing in Rb-/- p130-/- mice and compared this to murine Rb-/- p107-/- tumors and human tumors. Chimeric mice were made by injection of 129/Ola-derived Rb-/- p130-/- embryonic stem cells into wild type C57BL/6 blastocysts. SCNAs of retinoblastoma samples were determined by low-coverage (∼0.5×) whole genome sequencing. In Rb-/- p130-/- tumors, SCNAs included gain of chromosomes 1 (3/23 tumors), 8 (1/23 tumors), 10 (1/23 tumors), 11 (2/23 tumors), and 12 (4/23 tumors), which could be mapped to frequently altered chromosomes in human retinoblastomas. While the altered chromosomes in Rb-/- p130-/- tumors were similar to those in Rb-/- p107-/- tumors, the alteration frequencies were much lower in Rb-/- p130-/- tumors. Most of the Rb-/- p130-/- tumors (16/23 tumors, 70%) were devoid of SCNAs, in strong contrast to Rb-/- p107-/- tumors, which were never (0/15 tumors) SCNA-devoid. Similarly, to human retinoblastoma, increased age at diagnosis significantly correlated with increased SCNA frequencies. Additionally, focal loss of Cdh11 was observed in one Rb-/- p130-/- tumor, which enforces studies in human retinoblastoma that identified CDH11 as a retinoblastoma suppressor. Moreover, based on a comparison of genes altered in human and murine retinoblastoma, we suggest exploring the role of HMGA1 and SRSF3 in retinoblastoma development. © 2016 Wiley Periodicals, Inc.

The DREAM complex in antitumor activity of imatinib mesylate in gastrointestinal stromal tumors.

PURPOSE OF REVIEW: Although most gastrointestinal stromal tumors respond well to treatment with the small molecule kinase inhibitor imatinib mesylate (Gleevec), complete remissions are rare and the majority of patients achieve disease stabilization. Furthermore, discontinuation of treatment in the presence of residual tumor mass almost inevitably leads to tumor progression. These observations suggest that a subset of tumor cells not only persists under imatinib treatment, but remains viable. The current article reviews the molecular basis for these findings and explores strategies to exploit them therapeutically. RECENT FINDINGS: Although imatinib induces apoptosis in a subset of gastrointestinal stromal tumor cells, it leads to a reversible exit from the cell division cycle and entry into G0, a cell cycle state called quiescence, in the remaining cells. Mechanistically, this process involves the DREAM complex (DP, p130/RBL2, E2F4 and MuvB), a newly identified key regulator of quiescence. Interfering with DREAM complex formation either by siRNA-mediated knockdown or by pharmacological inhibition of the regulatory kinase dual-specificity tyrosine phosphorylation-regulated kinase 1A was shown to enhance imatinib-induced gastrointestinal stromal tumor cell death. SUMMARY: Targeting the DREAM complex and imatinib-induced quiescence could provide opportunities for future therapeutic interventions toward more efficient imatinib responses.

Loss of p130 accelerates tumor development in a mouse model for human small-cell lung carcinoma.

Small-cell lung carcinoma (SCLC) is a neuroendocrine subtype of lung cancer. Although SCLC patients often initially respond to therapy, tumors nearly always recur, resulting in a 5-year survival rate of less than 10%. A mouse model has been developed based on the fact that the RB and p53 tumor suppressor genes are mutated in more than 90% of human SCLCs. Emerging evidence in patients and mouse models suggests that p130, a gene related to RB, may act as a tumor suppressor in SCLC cells. To test this idea, we used conditional mutant mice to delete p130 in combination with Rb and p53 in adult lung epithelial cells. We found that loss of p130 resulted in increased proliferation and significant acceleration of SCLC development in this triple-knockout mouse model. The histopathologic features of the triple-mutant mouse tumors closely resembled that of human SCLC. Genome-wide expression profiling experiments further showed that Rb/p53/p130-mutant mouse tumors were similar to human SCLC. These findings indicate that p130 plays a key tumor suppressor role in SCLC. Rb/p53/p130-mutant mice provide a novel preclinical mouse model to identify novel therapeutic targets against SCLC.

Senescence and p130/Rbl2: a new beginning to the end.

Senescence is the process of cellular aging dependent on the normal physiological functions of non-immortalized cells. With increasing data being uncovered in this field, the complex molecular web regulating senescence is gradually being unraveled. Recent studies have suggested two main phases of senescence, the triggering of senescence and the maintenance of senescence. Each has been supported by data implying precise roles for DNA methyltransferases, reactive oxygen species and other factors. We will first summarize the data supporting these claims and then highlight the specific role that we hypothesize that p130/Rbl2 plays in the modulation of the senescence process.

Cell cycle kinases as therapeutic targets for cancer.

Several families of protein kinases orchestrate the complex events that drive the cell cycle, and their activity is frequently deregulated in hyperproliferative cancer cells. Although several molecules that inhibit cell cycle kinases have been developed and clinically screened as potential anticancer agents, none of these has been approved for commercial use and an effective strategy to specifically control malignant cell proliferation has yet to be established. However, recent genetic and biochemical studies have provided information about the requirement for certain cell cycle kinases by specific tumours and specialized tissue types. Here, we discuss the potential and limitations of established cell cycle kinases as targets in anticancer drug discovery as well as novel strategies for the design of new agents.

The retinoblastoma protein and its homolog p130 regulate the G1/S transition in pancreatic beta-cells.

OBJECTIVE: The retinoblastoma protein family (pRb, p130, p107) plays a central role in the regulation of cell cycle progression. Surprisingly, loss of pRb in the beta-cell has no discernible effect on cell cycle control. Therefore, we explored the effects of individual loss of either p130 or p107 in addition to the simultaneous loss of both pRb/p130 on the beta-cell. RESEARCH DESIGN AND METHODS: Adult mice deficient in either p130 or p107 or both pRb/p130 were examined for effects on beta-cell replication, function, and survival. The Cre-Lox system was also used to inactivate pRb in wild-type and p130-deficient beta-cells in vitro. RESULTS: In vivo loss of either p107 or p130 did not affect beta-cell replication or function. Combined pRb/p130 loss, however, resulted in dramatically accelerated proliferation as well as apoptotic cell death. Pancreas and beta-cell mass were significantly reduced in double mutants. Despite this, overall glucose tolerance was normal, except for mild postprandial hyperglycemia. Ex vivo, acute deletion of pRb in p130-deficient beta-cells also caused a striking increase in proliferation. The combined deletion of pRb/p130 upregulated islet expression of E2F2 but not E2F1. CONCLUSIONS: These studies define an essential role for the pocket proteins in controlling the G(1)/S transition in beta-cells. When deficient in both pRb and p130, beta-cells undergo unrestrained cell cycle reentry and activation of apoptosis. These studies underscore the central role of the pRb pathway in controlling beta-cell turnover and provide new cellular targets for beta-cell regeneration.

The retinoblastoma gene Rb and its family member p130 suppress lung adenocarcinoma induced by oncogenic K-Ras.

Mutations of the retinoblastoma tumor suppressor gene RB are frequently observed in human cancers, but rarely in non-small cell lung carcinomas (NSCLCs). Emerging evidence also suggests that the RB-related gene p130 is inactivated in a subset of human NSCLCs. To directly test the specific tumor suppressor roles of RB and p130 in NSCLC, we crossed Rb and p130 conditional mutant mice to mice carrying a conditional oncogenic K-Ras allele. In this model, controlled oncogenic K-Ras activation leads to the development of adenocarcinoma, a major subtype of NSCLC. We found that loss of p130 accelerated the death of mice, providing direct evidence in vivo that p130 is a tumor suppressor gene, albeit a weak one in this context. Loss of Rb increased the efficiency of lung cancer initiation and resulted in the development of high-grade adenocarcinomas and rapid death. Thus, despite the low frequency of RB mutations in human NSCLCs and reports that K-Ras activation and loss of RB function are rarely found in the same human tumors, loss of Rb clearly cooperates with activation of oncogenic K-Ras in lung adenocarcinoma development in mice.

The retinoblastoma family member pRb2/p130 is an independent predictor of survival in human soft tissue sarcomas.

PURPOSE: pRb2/p130, a member of the Retinoblastoma gene family, has been shown to be a powerful prognostic factor in several malignancies. We sought to evaluate pRb2/p130 protein expression and its clinical effect in patients affected with soft tissue sarcomas (STS). EXPERIMENTAL DESIGN: Expression of pRb2/p130 was evaluated by immunohistochemistry on formalin-fixed, paraffin-embedded sections in 41 STSs. Results obtained were correlated with clinicopathologic variables and disease-free and overall survival (OS) in univariate and multivariate analysis. RESULTS: Expression of pRb2/p130 was diminished in 25 (61%) tumors, whereas the remaining ones (39%) were classified as high expressors. No correlation between pRb2/p130 expression and clinicopathologic variables was observed. However, a direct relationship between pRb2/p130 expression and clinical outcome of the patients was found in the subgroup of nonmetastatic tumors (n = 31). In univariate analysis, reduced pRb2/p130 expression was a negative prognostic factor and correlated with shorter disease-free survival (P = 0.021) and OS (P = 0.017) survival. In multivariate analysis, reduced pRb2/p130 expression was confirmed to be an independent predictor of shorter OS when considered together with tumor stage and grading (risk ratio, 7.893; confidence interval, 1.618-38.509; P = 0.011). CONCLUSIONS: This study shows for the first time the potential prognostic value of pRb2/130 expression evaluated on formalin-fixed, paraffin-embedded sections in STSs patients. pRb2/p130 immunoreactivity can be used to predict OS in patients with nonmetastatic STSs and, therefore, may represent a new prognostic marker.

Novel p53/p130 axis in bladder tumors.

OBJECTIVES: To identify the relationships between key components of the proliferative and apoptotic pathways in bladder tumors. METHODS: A tissue array of 88 bladder tumors was assembled. Immunohistochemical analyses were used to investigate the relationship between nine different parameters: stage, proliferation (Ki67), apoptosis (in situ DNA nick end labeling), the anti-apoptotic protein Bcl-2, tumor suppressors p53 and retinoblastoma protein (Rb), the Rb-related protein p130, cyclin E, and the cyclin-dependent kinase inhibitor p27. The protein expression in each tumor was reported as the percentage of positively staining cells. RESULTS: The analysis focused on Stage 1 to 3 tumors. Analysis found that p53 expression increased progressively with stage, and Rb and p27 decreased with increasing stage. Overall, the cyclin E levels correlated with the proliferative index. Cyclin E levels were low in Stage 1 tumors and elevated in Stage 2 tumors, but were decreased in Stage 3 tumors. Multivariate analysis uncovered a correlation between cyclin E and proliferation (Ki67) and a weak correlation between p53 and Bcl-2 and between p27 and Rb. A strong correlation was found between the expression of p53 and p130, which was apparent in Stages 1 and 3, but not in Stage 2. Furthermore, high levels of p130 protein were detected primarily in the cytoplasm. CONCLUSIONS: These results suggest a novel p53/p130 axis in bladder tumors.

pRB family proteins are required for H3K27 trimethylation and Polycomb repression complexes binding to and silencing p16INK4alpha tumor suppressor gene.

Genetic studies have demonstrated that Bmi1 promotes cell proliferation and stem cell self-renewal with a correlative decrease of p16(INK4a) expression. Here, we demonstrate that Polycomb genes EZH2 and BMI1 repress p16 expression in human and mouse primary cells, but not in cells deficient for pRB protein function. The p16 locus is H3K27-methylated and bound by BMI1, RING2, and SUZ12. Inactivation of pRB family proteins abolishes H3K27 methylation and disrupts BMI1, RING2, and SUZ12 binding to the p16 locus. These results suggest a model in which pRB proteins recruit PRC2 to trimethylate p16, priming the BMI1-containing PRC1L ubiquitin ligase complex to silence p16.

A small molecule based on the pRb2/p130 spacer domain leads to inhibition of cdk2 activity, cell cycle arrest and tumor growth reduction in vivo.

One strategy in the development of anticancer therapeutics has been to arrest malignant proliferation through inhibition of the enzymatic activity of cyclin-dependent kinases (cdks), which are key regulatory molecules of the cell cycle. Over the past few years, numerous compounds with remarkable cdk inhibitory activity have been studied in cancer therapy, although it is very difficult to point out the best cdk to target. An excellent candidate appears to be cdk2, whose alteration is a pathogenic hallmark of tumorigenesis. The small molecule described in our study showed an inhibitory effect on the kinase activity of cdk2, a significant growth arrest observed in a colony formation assay and a reduction in the size of the tumor in nude mice, thus suggesting its potential role as a promising new type of mechanism-based antitumor drug, also for the treatment of hyperproliferative disorders.

Regulation of cellular senescence by Rb2/p130.

Growth regulatory functions of Rb2/p130, which aim at a sustained arrest such as in quiescent or differentiated cells, qualify the protein also to act as a central regulator of growth arrest in cellular senescence. In this respect, Rb2/p130 functions are connected to signaling pathways induced by p53, which is a master regulator in cellular senescence. Here, we summarize the pathways, which specify pRb2/p130 to control this arrest program and distinguish its functions from those of pRb/p105.

Rb2/p130 and protein phosphatase 2A: key mediators of ovarian carcinoma cell growth suppression by all-trans retinoic acid.

Despite a number of attempts to improve treatment of ovarian cancer, it remains the most common cause of death from gynecological cancers. Thus, it is very important to identify more effective drugs for treatment and prevention of ovarian cancer. All-trans-retinoic acid (ATRA) has been shown to arrest the growth of ovarian carcinoma cells in G0/G1 and to significantly elevate levels of Rb2/p130 protein, a member of the retinoblastoma family of tumor suppressors. As ATRA treatment leads to a significant increase in the amount of Rb2/p130 protein but not mRNA, the elevated levels of Rb2/p130 protein is likely the result of increased stability. In studies to elucidate the mechanism by which ATRA alters Rb2/p130 stability in ovarian cancer cells, it was determined that PP2A, a serine/threonine phosphatase, binds and dephosphorylates Rb2/p130. Dephosphorylated Rb2/p130 exhibits decreased ubiquitination and thus is not degraded by the proteasome. The sites at which PP2A catalytic subunit (PP2Ac) interacts with Rb2/p130 have been localized to the NLS in the C-terminus of Rb2/p130. These sites are also involved in the interaction of Rb/p130 with importin beta and importin alpha, members of the nuclear transport machinery. It is known that importin alpha recognizes a NLS on a target protein and importin beta binds the nuclear pore complex. Moreover, it has been shown that the binding of importin alpha to NLS significantly decreases with phosphorylation of NLS. In ATRA-treated ovarian carcinoma cells, PP2A binds to Rb2/p130 and dephosphorylates the NLS of Rb2/p130 leading to the interaction of importin alpha with Rb2/p130. Importin beta then binds to the importin alpha-Rb2/p130 complex, leading to the translocation of the Rb2/p130 to the nucleus where it acts to arrest ovarian cancer cells in G1 and suppress proliferation.

Pocket protein function in melanocyte homeostasis and neoplasia.

Melanoma is the most lethal of human skin cancers and its incidence is increasing worldwide [L.K. Dennis (1999). Arch. Dermatol. 135, 275; C. Garbe et al. (2000). Cancer 89, 1269]. Melanomas often metastasize early during the course of the disease and are then highly intractable to current therapeutic regimens [M.F. Demierre and G. Merlino (2004). Curr. Oncol. Rep. 6, 406]. Consequently, understanding the factors that maintain melanocyte homeostasis and prevent their neoplastic transformation into melanoma is of utmost interest from the perspective of therapeutic interdiction. This review will focus on the role of the pocket proteins (PPs), Rb1 (retinoblastoma protein), retinoblastoma-like 1 (Rbl1 also known as p107) and retinoblastoma-like 2 (Rbl2 also known as p130), in melanocyte homeostasis, with particular emphasis on their functions in the cell cycle and the DNA damage repair response. The potential mechanisms of PP deregulation in melanoma and the possibility of PP-independent pathways to melanoma development will also be considered. Finally, the role of the PP family in ultraviolet radiation (UVR)-induced melanoma and the precise contribution that each PP family member makes to melanocyte homeostasis will be discussed in the context of a number of genetically engineered mouse models.

The Rb-related p130 protein controls telomere lengthening through an interaction with a Rad50-interacting protein, RINT-1.

The oncogenic process often leads to a loss of normal telomere length control, usually as a result of activation of telomerase. Nevertheless, there are also telomerase-independent events that involve a Rad50-dependent recombination mechanism to maintain telomere length. Previous work has implicated the Rb family of proteins in the control of telomere length, and we now demonstrate that the p130 member of the Rb family is critical for telomere length control. p130 interacts specifically with the RINT-1 protein, previously identified as a Rad50-interacting protein. We further show that RINT-1 is essential for telomere length control. We propose that p130, forming a complex with Rad50 through RINT-1, blocks telomerase-independent telomere lengthening in normal cells. Given previous work implicating E2F in the control of telomerase gene expression, these results thus point to complementary roles for the Rb/E2F pathway in the control of telomere length.

Interaction of PP2A catalytic subunit with Rb2/p130 is required for all-trans retinoic acid suppression of ovarian carcinoma cell growth.

All-trans retinoic acid (ATRA) treatment causes CAOV3 ovarian carcinoma cells to growth arrest in the G0/G1 phase and to elevate the level of Rb2/p130 protein. PP2A, a serine/threonine phosphatase, binds and dephosphorylates Rb2/p130, thereby increasing the half-life of Rb2/p130 in the cell. In order to further characterize the interaction between Rb2/p130 and PP2A upon ATRA treatment, we examined the posttranslational modification of PP2A. ATRA treatment leads to hypophosphorylation of PP2A catalytic subunit (PP2Ac) that correlates with increased PP2A activity. In addition, the N-terminus of PP2Ac binds directly to NLS sequences located in the C-terminus of Rb2/p130. Furthermore, CAOV3 cells transfected with a truncated Rb2/p130 construct consisting of only the wt C-terminus grew more aggressively and were less sensitive to ATRA treatment when compared to parental CAOV3 cells. In contrast, CAOV3 cells transfected with a truncated Rb2/p130 construct consisting of only the C-terminus in which the NLS sites were mutated and which could not interact with PP2A, were as sensitive to ATRA treatment as parental CAOV3 cells. These studies suggest that ATRA treatment suppresses the growth of CAOV3 cells via a novel posttranscriptional mechanism involving PP2A.

Mitogen requirement for cell cycle progression in the absence of pocket protein activity.

Primary mouse embryonic fibroblasts lacking expression of all three retinoblastoma protein family members (TKO MEFs) have lost the G1 restriction point. However, in the absence of mitogens these cells become highly sensitive to apoptosis. Here, we show that TKO MEFs that survive serum depletion pass G1 but completely arrest in G2. p21CIP1 and p27KIP1 inhibit Cyclin A-Cdk2 activity and sequester Cyclin B1-Cdk1 in inactive complexes in the nucleus. This response is alleviated by mitogen restimulation or inactivation of p53. Thus, our results disclose a cell cycle arrest mechanism in G2 that restricts the proliferative capacity of mitogen-deprived cells that have lost the G1 restriction point. The involvement of p53 provides a rationale for the synergism between loss of Rb and p53 in tumorigenesis.