Regulation of transcription and chromatin structure by pRB: Here, there and everywhere

Commitment to divide is one of the most crucial steps in the mammalian cell division cycle. It is critical for tissue and organismal homeostasis, and consequently is highly regulated. The vast majority of cancers evade proliferative control, further emphasizing the importance of the commitment step in cell cycle regulation. The Retinoblastoma (RB) tumor suppressor pathway regulates this decision-making step. Since being the subject of Knudson\u27s \u27two hit hypothesis\u27, there has been considerable interest in understanding pRB\u27s role in cancer. It is best known for repressing E2F dependent transcription of cell cycle genes. However, pRB\u27s role in controlling chromatin structure is expanding and bringing it into new regulatory paradigms. In this review we discuss pRB function through protein-protein interactions, at the level of transcriptional regulation of individual promoters and in organizing higher order chromatin domains. © 2012 Landes Bioscience

The retinoblastoma protein and PML collaborate to organize heterochromatin and silence E2F-responsive genes during senescence

Cellular senescence is characterized by silencing of genes involved in DNA replication and cell cycle progression. Stable repression is crucial for preventing inappropriate DNA synthesis and the maintenance of a prolonged senescent state. Many of these genes are targets for E2F transcription factors. The pRB pathway plays a major role in senescence by directly repressing E2Fs and also by regulating chromatin at the promoters of E2F target genes using its LXCXE cleft-dependent interactions. In this study, we sought to investigate the mechanisms by which pRB stably silences E2F target gene transcription during cellular senescence. We report that in mouse embryonic fibroblasts, endogenous promyelocytic leukemia protein (PML) associates with E2F target genes in a pRB LXCXE-dependent manner during HrasV12-induced senescence. Furthermore, using a PML-IV-induced senescence model, we show that the pRB LXCXE binding cleft is essential for PML association with gene promoters, silencing of E2F target genes, and stable cell cycle exit. Binding assays show that pRB can interact with PML specifically during senescence, suggesting that signaling events in senescence regulate assembly of PML and pRB to establish heterochromatin and create a permanent cell cycle arrest. © 2014 Landes Bioscience

Mutation of the LXCXE Binding Cleft of pRb Facilitates Transformation by ras In Vitro but Does Not Promote Tumorigenesis In Vivo

Background:The Retinoblastoma protein (pRB) is a key tumor suppressor that is functionally inactivated in most cancers. pRB regulates the cell division cycle and cell cycle exit through protein-protein interactions mediated by its multiple binding interfaces. The LXCXE binding cleft region of pRB mediates interactions with cellular proteins that have chromatin regulatory functions. Chromatin regulation mediated by pRB is required for a stress responsive cell cycle arrest, including oncogene induced senescence. The in vivo role of chromatin regulation by pRB during senescence, and its relevance to cancer is not clear.Methodology/Principal Findings:Using gene-targeted mice, uniquely defective for pRB mediated chromatin regulation, we investigated its role during transformation and tumor progression in response to activation of oncogenic ras. We report that the pRBΔL mutation confers susceptibility to escape from HrasV12 induced senescence and allows transformation in vitro, although these cells possess high levels of DNA damage. Intriguingly, LSL-Kras, Rb1ΔL/ΔL mice show delayed lung tumor formation compared to controls. This is likely due to the increased apoptosis seen in the early hyperplastic lesions shortly following ras activation that inhibits tumor progression. Furthermore, DMBA treatment to induce sporadic ras mutations in other tissues also failed to reveal greater susceptibility to cancer in Rb1ΔL/ΔL mice.Conclusions/Significance:Our data suggests that chromatin regulation by pRB can function to limit proliferation, but its loss fails to contribute to cancer susceptibility in ras driven tumor models because of elevated levels of DNA damage and apoptosis. © 2013 Talluri et al

Multiple molecular interactions redundantly contribute to RB-mediated cell cycle control

Background: The G1-S phase transition is critical to maintaining proliferative control and preventing carcinogenesis. The retinoblastoma tumor suppressor is a key regulator of this step in the cell cycle. Results: Here we use a structure-function approach to evaluate the contributions of multiple protein interaction surfaces on pRB towards cell cycle regulation. SAOS2 cell cycle arrest assays showed that disruption of three separate binding surfaces were necessary to inhibit pRB-mediated cell cycle control. Surprisingly, mutation of some interaction surfaces had no effect on their own. Rather, they only contributed to cell cycle arrest in the absence of other pRB dependent arrest functions. Specifically, our data shows that pRB-E2F interactions are competitive with pRB-CDH1 interactions, implying that interchangeable growth arrest functions underlie pRB\u27s ability to block proliferation. Additionally, disruption of similar cell cycle control mechanisms in genetically modified mutant mice results in ectopic DNA synthesis in the liver. Conclusions: Our work demonstrates that pRB utilizes a network of mechanisms to prevent cell cycle entry. This has important implications for the use of new CDK4/6 inhibitors that aim to activate this proliferative control network

A retinoblastoma allele that is mutated at its common E2F interaction site inhibits cell proliferation in gene-targeted mice

The retinoblastoma protein (pRB) is best known for regulating cell proliferation through E2F transcription factors. In this report, we investigate the properties of a targeted mutation that disrupts pRB interactions with the transactivation domain of E2Fs. Mice that carry this mutation endogenously (Rb1δG) are defective for pRB-dependent repression of E2F target genes. Except for an accelerated entry into S phase in response to serum stimulation, cell cycle regulation in Rb1δG/δG mouse embryonic fibroblasts (MEFs) strongly resembles that of the wild type. In a serum deprivation-induced cell cycle exit, Rb1δG/δG MEFs display a magnitude of E2F target gene derepression similar to that of Rb1-/- cells, even though Rb1δG/δG cells exit the cell cycle normally. Interestingly, cell cycle arrest in Rb1δG/δG MEFs is responsive to p16 expression and gamma irradiation, indicating that alternate mechanisms can be activated in G1 to arrest proliferation. Some Rb1δG/δG mice die neonatally with a muscle degeneration phenotype, while the others live a normal life span with no evidence of spontaneous tumor formation. Most tissues appear histologically normal while being accompanied by derepression of pRB-regulated E2F targets. This suggests that non- E2F-, pRB-dependent pathways may have a more relevant role in proliferative control than previously identified. © 2014, American Society for Microbiology

Integrated genomics and comprehensive validation reveal drivers of genomic evolution in esophageal adenocarcinoma

Esophageal adenocarcinoma (EAC) is associated with a marked genomic instability, which underlies disease progression and development of resistance to treatment. In this study, we used an integrated genomics approach to identify a genomic instability signature. Here we show that elevated expression of this signature correlates with poor survival in EAC as well as three other cancers. Knockout and overexpression screens establish the relevance of these genes to genomic instability. Indepth evaluation of three genes (TTK, TPX2 and RAD54B) confirms their role in genomic instability and tumor growth. Mutational signatures identified by whole genome sequencing and functional studies demonstrate that DNA damage and homologous recombination are common mechanisms of genomic instability induced by these genes. Our data suggest that the inhibitors of TTK and possibly other genes identified in this study have potential to inhibit/reduce growth and spontaneous as well as chemotherapy-induced genomic instability in EAC and possibly other cancers