Interchangeable Roles for E2F Transcriptional Repression by the Retinoblastoma Protein and p27KIP1–Cyclin-Dependent Kinase Regulation in Cell Cycle Control and Tumor Suppression
Abstract:The mammalian G 1 -S phase transition is controlled by the opposing forces of cyclin-dependent kinases (CDK) and the retinoblastoma protein (pRB). Here, we present evidence for systems-level control of cell cycle arrest by pRB-E2F and p27-CDK regulation. By introducing a point mutant allele of pRB that is defective for E2F repression (Rb1 G ) into a p27 KIP1 null background (Cdkn1b Ϫ/Ϫ ), both E2F transcriptional repression and CDK regulation are compromised. These double-mutant Rb1 G/G ; Cdkn1b Ϫ/Ϫ mice are v… Show more
“…This experiment suggests that pRB is unable to engage E2F3 and CDH1 dependent functions simultaneously, suggesting that these functions are interchangeable. This mirrors findings from recent in vivo approaches to pRB dependent cell cycle control [30], and this will be explored further in the discussion.Fig. 3Competition between E2Fs and CDH1 for pRB binding.…”
BackgroundThe 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.ResultsHere 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’s 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.ConclusionsOur 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.
“…This experiment suggests that pRB is unable to engage E2F3 and CDH1 dependent functions simultaneously, suggesting that these functions are interchangeable. This mirrors findings from recent in vivo approaches to pRB dependent cell cycle control [30], and this will be explored further in the discussion.Fig. 3Competition between E2Fs and CDH1 for pRB binding.…”
BackgroundThe 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.ResultsHere 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’s 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.ConclusionsOur 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.
“…We have shown that the Rb1 G mutation exacerbates cancer development in Trp53 −/− mice, but has essentially no effect on tumor-free survival in the presence of oncogenic Kras G12D , or from loss of p21 ( Cdkn1a −/− ). In conjunction with prior results that Rb1 G/G ; Cdkn1b −/− animals succumb to pituitary tumors (18), and that Rb1 +/− ; Cdkn1a −/− mice have accelerated pituitary tumorigenesis (29), these genetic crosses provide two simple conclusions relating to RB function in cell cycle and cancer susceptibility. The first is that compromised RB-E2F transcriptional regulation is not uniformly cancer promoting, even though it potently contributes in some crosses.…”
Section: Discussionsupporting
confidence: 53%
“…3D). This is an important observation as Rb1 +/− ; Cdkn1a −/− mice are susceptible to pituitary tumors and have similar survival as for Rb1 G/+ ; Cdkn1b −/− animals (18, 29), this indicates that the Rb1 G mutation cooperates preferentially with p27 loss.…”
20 21 RB-E2F transcriptional control plays a key role in regulating the timing of cell cycle 22 progression from G1 to S-phase in response to growth factor stimulation. Despite this 23 role, it is genetically dispensable for cell cycle exit in primary fibroblasts in response to 24 growth arrest signals. Mice engineered to be defective for RB-E2F transcriptional 25 control at cell cycle genes were also found to live a full lifespan with no susceptibility to 26 cancer. Based on this background we sought to probe the vulnerabilities of RB-E2F 27 transcriptional control defects found in Rb1 R461E,K542E mutant mice (Rb1 G ) through genetic 28 crosses with other mouse strains. We generated Rb1 G/G mice in combination with Trp53 29 and Cdkn1a deficiencies, as well as in combination with Kras G12D . The Rb1 G mutation 30 enhanced Trp53 cancer susceptibility, but had no effect in combination with Cdkn1a 31 deficiency or Kras G12D . Collectively, this study indicates that compromised RB-E2F 32 transcriptional control is not uniformly cancer enabling, but rather has potent oncogenic 33 effects when combined with specific vulnerabilities.
35 363 37 Introduction 38 39The maintenance of cell cycle control is crucial to the normal development and 40 homeostasis of multicellular organisms (1). In addition, misregulation of the cell cycle is 41 widespread in tumorigenesis (2). To ensure that cells only replicate their genome once 42 per cell cycle, the regulation of G1 to S-phase is tightly controlled (3). At the core of G1-43 S regulation are Cyclin dependent kinases (CDKs) and the Retinoblastoma (RB) family 44 of proteins. Proliferative signals generally activate Ras and lead to Cyclin D-CDK4 or 6 45 upregulation, phosphorylation of RB, and the release of activator E2F transcription 46 factors to induce cell cycle entry (4). This is complemented by CDK phosphorylation of 47 the RB family protein p130 that disassembles the DREAM transcriptional repressor 48 complex, further contributing to E2F activation in early G1 (5). In addition, Cyclin E-49 CDK2 is negatively regulated by the CDK inhibitor protein p27 in late G1 and its 50 degradation coincides with maximal CDK2 activation and the commitment to S-phase 51 entry (6). Thus, both CDKs and RB family members are key to the commitment step to 52 enter the cell cycle and over expression of G1 Cyclin-CDKs accelerates entry into S-53 phase, as does loss of RB, or the combination of its family members p107 and p130 (7-9).54 While CDK and E2F regulation are well known in cell cycle control, emerging roles in 55 cell lineage commitment suggest that RB-E2F transcription may serve more purposes 56 than just cell cycle entry decisions, as it is only one piece of a complex E2F 57 transcriptional network that operates in the G1 phase (10).
58In addition to regulating entry into the cell cycle, many of the same molecules 59 function to execute a transient cell cycle arrest, or more permanent cell cycle exit 60 decisions. For example, DNA damage stabilizes p53 and leads to transcriptional 4 6...
“…The exact roles of the individual E2F homologues in cancer progression are not fully understood. E2F1-E2F3 are the central regulators of cell cycle [56]. The activities are negatively regulated via binding with retinoblastoma-like 1 protein (RB; encoded by RBL1).…”
Background: Tumors are heterogeneous in nature, composed of different cell populations with various mutations and/or phenotypes. Using a single drug to encounter cancer progression is generally ineffective. To improve the treatment outcome, multiple drugs of distinctive mechanisms but complementary anticancer activities (combination therapy) are often used to enhance antitumor efficacy and minimize the risk of acquiring drug resistance. We report here the synergistic effects of salinomycin (a polyether antibiotic) and dasatinib (a Src kinase inhibitor). Methods: Functionally, both drugs induce cell cycle arrest, intracellular reactive oxygen species (iROS) production, and apoptosis. We rationalized that an overlapping of the drug activities should offer an enhanced anticancer effect, either through vertical inhibition of the Src-STAT3 axis or horizontal suppression of multiple pathways. We determined the toxicity induced by the drug combination and studied the kinetics of iROS production by fluorescence imaging and flow cytometry. Using genomic and proteomic techniques, including RNA-sequencing (RNA-seq), reverse transcriptionquantitative polymerase chain reaction (RT-qPCR), and Western Blot, we subsequently identified the responsible pathways that contributed to the synergistic effects of the drug combination. Results: Compared to either drug alone, the drug combination showed enhanced potency against MDA-MB-468, MDA-MB-231, and MCF-7 human breast cancer (BC) cell lines and tumor spheroids. The drug combination induces both iROS generation and apoptosis in a time-dependent manner, following a 2-step kinetic profile. RNA-seq data revealed that the drug combination exhibited synergism through horizontal suppression of multiple pathways, possibly through a promotion of cell cycle arrest at the G1/S phase via the estrogen-mediated S-phase entry pathway, and partially via the BRCA1 and DNA damage response pathway.
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