Yihong Ma

Interactions between E2F1 and SirT1 regulate apoptotic response to DNA damage

The nicotinamide adenine dinucleotide (NAD)-dependent deacetylase Sir2 (silent information regulator 2) regulates gene silencing in yeast and promotes lifespan extension during caloric restriction. The mammalian homologue of Sir2 (SirT1) regulates p53, NF-κB and Forkhead transcription factors, and is implicated in stress response. This report shows that the cell-cycle and apoptosis regulator E2F1 induces SirT1 expression at the transcriptional level. Furthermore, SirT1 binds to E2F1 and inhibits E2F1 activities, forming a negative feedback loop. Knockdown of SirT1 by small interference RNA (siRNA) increases E2F1 transcriptional and apoptotic functions. DNA damage by etoposide causes E2F1-dependent induction of SirT1 expression and knockdown of SirT1 increases sensitivity to etoposide. These results reveal a mutual regulation between E2F1 and SirT1 that affects cellular sensitivity to DNA damage.

Role of Stat3 in Regulating p53 Expression and Function

ABSTRACT Loss of p53 function by mutation is common in cancer. However, most natural p53 mutations occur at a late stage in tumor development, and many clinically detectable cancers have reduced p53 expression but no p53 mutations. It remains to be fully determined what mechanisms disable p53 during malignant initiation and in cancers without mutations that directly affect p53. We show here that oncogenic signaling pathways inhibit the p53 gene transcription rate through a mechanism involving Stat3, which binds to the p53 promoter in vitro and in vivo. Site-specific mutation of a Stat3 DNA-binding site in the p53 promoter partially abrogates Stat3-induced inhibition. Stat3 activity also influences p53 response genes and affects UV-induced cell growth arrest in normal cells. Furthermore, blocking Stat3 in cancer cells up-regulates expression of p53, leading to p53-mediated tumor cell apoptosis. As a point of convergence for many oncogenic signaling pathways, Stat3 is constitutively activated at high frequency in a wide diversity of cancers and is a promising molecular target for cancer therapy. Thus, repression of p53 expression by Stat3 is likely to have an important role in development of tumors, and targeting Stat3 represents a novel therapeutic approach for p53 reactivation in many cancers lacking p53 mutations.

Direct repression of the Mcl-1 promoter by E2F1

E2F1 induces apoptosis via both p53-dependent and p53-independent mechanisms. The direct targets in the p53-independent pathway remain enigmatic; however, the induction of this pathway does not require the transactivation domain of E2F1. Using cells that are defective in p53 activation, we show that E2F1 potently represses the expression of Mcl-1 – an anti-apoptotic Bcl-2 family member whose depletion results in apoptosis. We also show that this transcriptional repression is direct and dependent upon E2F1s DNA-binding domain, but does not require the transactivation domain of E2F1. Consistent with this DNA binding requirement of E2F1 to repress Mcl-1, we show that E2F1 binds to the Mcl-1 promoter both in vitro and in vivo, and have identified the DNA element (−143/−117) within this promoter that is required for E2F1 binding and repression. Additionally, cell lines constitutively expressing Mcl-1 are resistant to E2F1-mediated apoptosis – suggesting that Mcl-1 downregulation is a necessary event in the p53-independent apoptotic process. Thus, we identify a p53 family-independent mechanism of E2F1-induced apoptosis in which E2F1 directly represses Mcl-1 expression.

Activation of p27Kip1 Expression by E2F1 A NEGATIVE FEEDBACK MECHANISM

The E2F1 transcription factor is a critical regulator of cell cycle due to its ability to promote S phase entry. However, E2F1 overexpression also sensitizes cells to apoptosis and E2F1-null mice are predisposed to tumor development, suggesting that it also has properties of a growth suppressor. E2F1 transcription function is regulated by interaction with hypophosphorylated pRb. Cdk inhibitors such as p16INK4a and p27Kip1 inhibit pRb phosphorylation by the cyclin D/Cdk4 and cyclin E/Cdk2 complexes, thus keeping E2F1 in an inactive state. We found that E2F1 binds to the p27 promoter in vivo and activates p27 mRNA and protein expression. Depletion of endogenous E2F1 by siRNA causes a reduction in basal p27 expression level. Inhibition of endogenous p27 expression by siRNA increases E2F1 transcriptional activity and permits accelerated cell cycle progression by exogenous E2F1. These observations suggest that induction of p27 acts as a negative feedback mechanism for E2F1 and may also contribute to other functions of E2F1.

Histone Deacetylase Inhibitors Downregulate Checkpoint Kinase 1 Expression to Induce Cell Death in Non-Small Cell Lung Cancer Cells

Background Histone deacetylase inhibitors (HDACis) are promising anticancer drugs; however, the molecular mechanisms leading to HDACi-induced cell death have not been well understood and no clear mechanism of resistance has been elucidated to explain limited efficacy of HDACis in clinical trials. Methods and Findings Here, we show that protein levels of checkpoint kinase 1 (Chk1), which has a major role in G2 cell cycle checkpoint regulation, was markedly reduced at the protein and transcriptional levels in lung cancer cells treated with pan-and selective HDACis LBH589, scriptaid, valproic acid, apicidin, and MS-275. In HDACi treated cells Chk1 function was impaired as determined by decreased inhibitory phosphorylation of cdc25c and its downstream target cdc2 and increased expression of cdc25A and phosphorylated histone H3, a marker of mitotic entry. In time course experiments, Chk1 downregulation occurred after HDACi treatment, preceding apoptosis. Ectopic expression of Chk1 overcame HDACi-induced cell death, and pretreating cells with the cdc2 inhibitor purvalanol A blocked entry into mitosis and prevented cell death by HDACis. Finally, pharmacological inhibition of Chk1 showed strong synergistic effect with LBH589 in lung cancer cells. Conclusions These results define a pathway through which Chk1 inhibition can mediate HDACi-induced mitotic entry and cell death and suggest that Chk1 could be an early pharmacodynamic marker to assess HDACi efficacy in clinical samples.

A Small-Molecule E2F Inhibitor Blocks Growth in a Melanoma Culture Model

HLM006474 was identified using a computer-based virtual screen and the known crystal structure of the DNA-bound E2F4/DP2 heterodimer. Treatment of multiple cell lines with HLM006474 resulted in the loss of intracellular E2F4 DNA-binding activity as measured by electrophoretic mobility shift assay within hours. Overnight exposure to HLM006474 resulted in down-regulation of total E2F4 protein as well as known E2F targets. The effects of HLM006474 treatment on different cell lines varied but included a reduction in cell proliferation and an increase in apoptosis. HLM006474 induced apoptosis in a manner distinct from cisplatin and doxorubicin. E2F4-null mouse embryonic fibroblasts were less sensitive than wild-type counterparts to the apoptosis-inducing activity of the compound, revealing its biological specificity. A375 cells were extremely sensitive to the apoptosis-inducing activity of the compound in two-dimensional culture, and HLM006474 was a potent inhibitor of melanocytes proliferation and subsequent invasion in a three-dimensional tissue culture model system. Together, these results suggest that interference with E2F activity using small molecules may have clinical application in cancer therapy.

Bok, Bcl-2 related ovarian killer, is cell cycle regulated and sensitizes to stress-induced apoptosis

Bok/Mtd (Bcl-2-related ovarian killer/Matador) is considered a pro-apoptotic member of the Bcl-2 family. Although identified in 1997, little is known about its biological role. We have previously demonstrated that Bok mRNA is up-regulated following E2F1 overexpression. In the current work, we demonstrate that Bok RNA is low in quiescent cells and rises upon serum stimulation. To determine the mechanism underlying this regulation, we cloned and characterized the mouse Bok promoter. We find that the mouse promoter contains a conserved E2F binding site (-43 to -49) and that a Bok promoter-driven luciferase reporter is activated by serum stimulation dependent on this site. Chromatin immunoprecipitation assays demonstrate that endogenous E2F1 and E2F3 associate with the Bok promoter in vivo. Surprisingly, we find that H1299 cells can stably express high levels of exogenous Bok protein. However, these cells are highly sensitive to chemotherapeutic drug treatment. Taken together these results demonstrate that Bok represents a cell cycle-regulated pro-apoptotic member of the Bcl-2 family, which may predispose growing cells to chemotherapeutic treatment.

E2F4 deficiency promotes drug-induced apoptosis.

E2F1 and E2F4 are known to have opposing roles in cell cycle control. In the present work, we examine the role of both E2F1 and E2F4 in apoptosis induced by three cyclindependent kinase inhibitors (roscovitine, BMS-387032, and flavopiridol) as well as by three established chemotherapeutic drugs (VP16, cisplatin and paclitaxel). We find that E2F4 levels are diminished following treatment with cyclin dependent kinase inhibitors (flavopiridol, roscovitine and BMS-387032) or with DNA damaging drugs (cisplatin and VP16). In contrast, each of these drugs induced E2F1. We find that mouse fibroblasts nullizygous for the E2F4 gene are more sensitive to apoptosis induced by roscovitine, flavopiridol, cisplatin, and VP16, whereas E2F1-deficient fibroblasts are less sensitive. Likewise, we find that RNAi-mediated reductions in E2F4 in human cancer cells results in increased drug sensitivity. Taken together, these results support a model in which E2F1 and E2F4 play opposing roles during drug-induced apoptosis.

Identification of Aurora-A as a Direct Target of E2F3 during G2/M Cell Cycle Progression

Aurora-A is a centrosome kinase and plays a pivotal role in G2/M cell cycle progression. Expression of Aurora-A is cell cycle-dependent. Levels of Aurora-A mRNA and protein are low in G1/S, accumulate during G2/M, and decrease rapidly after mitosis. Previous studies have shown regulation of the Aurora-A protein level during the cell cycle through the ubiquitin-proteasome pathway. However, the mechanism of transcriptional regulation of Aurora-A remains largely unknown. Here, we demonstrated that E2F3 modulates Aurora-A mRNA expression during the cell cycle. Ectopic expression of E2F3 induces Aurora-A expression. Stable knockdown of E2F3 decreases mRNA and protein levels of Aurora-A and delays G2/M entry. Further, E2F3 directly binds to Aurora-A promoter and stimulates the promoter activity. Deletion and mutation analyses of the Aurora-A promoter revealed that a region located 96-bp upstream of the transcription initiation site is critical for the activation of the promoter by E2F3. In addition, expression of E2F3 positively correlates with the protein level of Aurora-A in human ovarian cancer examined. These results indicate for the first time that Aurora-A is transcriptionally regulated by E2F3 during the cell cycle and that E2F3 is a causal factor for up-regulation of Aurora-A in a subset of human ovarian cancer. Thus, the E2F3-Aurora-A axis could be an important target for cancer intervention.

RhoBTB2 (DBC2) Is a Mitotic E2F1 Target Gene with a Novel Role in Apoptosis

We have identified the RhoBTB2 putative tumor suppressor gene as a direct target of the E2F1 transcription factor. Overexpression of E2F1 led to up-regulation of RhoBTB2 at the level of mRNA and protein. This also occurred during the induction of E2F1 activity in the presence of cycloheximide, thus indicating that RhoBTB2 is a direct target. RNAi-mediated knockdown of E2F1 resulted in decreased RhoBTB2 protein expression, demonstrating that RhoBTB2 is a physiological target of E2F1. Because E2F1 primarily serves to transcribe genes involved in cell cycle progression and apoptosis, we explored whether RhoBTB2 played roles in either of these processes. We found RhoBTB2 expression highly up-regulated during mitosis, which was partially dependent on the presence of E2F1. Furthermore, overexpression of RhoBTB2 induced a short term increase in cell cycle progression and proliferation, while long term expression had a negative effect on these processes. We similarly found RhoBTB2 up-regulated during drug-induced apoptosis, with this being primarily dependent on E2F1. Finally, we observed that knockdown of RhoBTB2 levels via siRNA delayed the onset of drug-induced apoptosis. Collectively, we describe RhoBTB2 as a novel direct target of E2F1 with roles in cell cycle and apoptosis.