E2F-regulated Genes Research Articles

The Tumor Suppressor Maspin Mediates E2F1-Induced Sensitivity of Cancer Cells to Chemotherapy

The E2F1 transcription factor is a critical downstream target of the tumor suppressor RB. When activated, E2F1 can induce cell proliferation and/or apoptosis. In addition, E2F1 overexpression sensitizes cancer cells to chemotherapeutic drugs. In a screen for genes that are regulated synergistically by E2F1 and chemotherapy in cancer cells, we identified the proapoptotic tumor suppressor gene maspin (mammary serine protease inhibitor) as a novel E2F1-regulated gene. In line with being an E2F-regulated gene, maspin expression is inhibited by short hairpin RNA directed against E2F1 and increases upon activation of endogenous E2F. Furthermore, maspin mRNA and protein levels are elevated upon activation of exogenous E2F1. Importantly, we show that E2F1-mediated upregulation of maspin is enhanced by chemotherapeutic drugs, and inhibition of maspin expression significantly impairs the ability of E2F1 to promote chemotherapy-induced apoptosis. Summarily, our data indicate that maspin is an important effector of E2F1-induced chemosensitization.

E1A Interacts with Two Opposing Transcriptional Pathways To Induce Quiescent Cells into S Phase

Despite data suggesting that the adenovirus E1A protein of 243 amino acids creates an S-phase environment in quiescent cells by overcoming the nucleosomal repression of E2F-regulated genes, the precise mechanisms underlying E1A's ability in this process have not yet been defined at the biochemical level. In this study, we show by kinetic analysis that E1A, as opposed to an E1A mutant failing to bind p130, can temporally eliminate corepressor complexes consisting of p130-E2F4 and HDAC1/2-mSin3B from the promoters of E2F-regulated genes in quiescent cells. Once the complexes are removed, the di-methylation of H3K9 at these promoters becomes dramatically diminished, and this in turn allows for the acetylation of H3K9/14 and the recruitment of activating E2F family members, which is then followed by the transcriptional activity of the E2F-regulated genes. Remarkably, although an E1A mutant that can no longer bind to a histone acetyltransferase (PCAF) is as capable as wild-type E1A in eliminating corepressor complexes and methyl groups from the promoters of these genes, it cannot mediate the acetylation of H3K9/14 or induce their transcription. These findings suggest that corepressors as well as coactivators are acted upon by E1A to derepress E2F-regulated genes in quiescent cells. Thus, our results highlight for the first time a functional relationship between E1A and two transcriptional pathways of differing functions for transitioning cells out of quiescence and into S phase.

C/EBPβΔuORF mice—a genetic model for uORF-mediated translational control in mammals

Upstream ORFs (uORFs) are translational control elements found predominantly in transcripts of key regulatory genes. No mammalian genetic model exists to experimentally validate the physiological relevance of uORF-regulated translation initiation. We report that mice deficient for the CCAAT/enhancer-binding protein beta (C/EBPbeta) uORF initiation codon fail to initiate translation of the autoantagonistic LIP (liver inhibitory protein) C/EBPbeta isoform. C/EBPbeta(DeltauORF) mice show hyperactivation of acute-phase response genes, persistent repression of E2F-regulated genes, delayed and blunted S-phase entry of hepatocytes after partial hepatectomy, and impaired osteoclast differentiation. These data and the widespread prevalence of uORFs in mammalian transcriptomes suggest a comprehensive role of uORF-regulated translation in (patho)physiology.

FBXL16 is a novel E2F1-regulated gene commonly upregulated in p16INK4A- and p14ARF-silenced HeLa cells

Two crucial cell cycle regulators, p16INK4A and p14ARF, are produced from the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene locus by alternative reading frames; these regulators act as tumor suppressors during tumorigenesis. However, the molecular events incidental to the acute functional loss of CDKN2A remain a critical issue. Two pivotal regulatory pathways of cell fate determination involving p16INK4A/retinoblastoma protein (pRb)/E2F1 and p14ARF/p53 interact tightly with each other; however, novel factors with an integral or overlapping role in these two pathways remain incompletely defined. To this end, we specifically decreased the expression of p16INK4A or p14ARF proteins using RNA interference (RNAi) in HeLa cells. Using a DNA microarray approach, we showed that several genes are commonly regulated in both p16INK4A and p14ARF knockdown cells, compared with control RNA-treated cells. We focused on the FBXL16 (F-box and leucine-rich repeat protein 16) gene, the expression of which was reproducibly upregulated in p16INK4A and p14ARF knockdown cells, as evaluated using RT-PCR. Interestingly, the promoter region of FBXL16 was shown to be upregulated by activator E2Fs. Finally, RNAi-mediated knockdown of FBXL16 increased the cell proliferation rate of HeLa cells. Together, our results illustrate a unique aspect of the interdependence between the p16INK4A/pRb/E2F1 and p14ARF/p53 pathways at a molecular level.

Functional interplay between E2F1 and chemotherapeutic drugs defines immediate E2F1 target genes crucial for cancer cell death.

The E2F1 transcription factor enhances apoptosis by DNA damage in tumors lacking p53. To elucidate the mechanism of a potential cooperation between E2F1 and chemotherapy, whole-genome microarrays of chemoresistant tumor cell lines were performed focusing on the identification of cooperation response genes (CRG). This gene class is defined by a synergistic expression response upon endogenous E2F1 activation and drug treatment. Cluster analysis revealed an expression pattern of CRGs similar to E2F1 mono-therapy, suggesting that chemotherapeutics enhance E2F1-dependent gene expression at the transcriptional level. Using this approach as a tool to explore E2F1-driven gene expression in response to anticancer drugs, we identified novel apoptosis genes such as the tumor suppressor TIEG1/KLF10 as direct E2F1 targets. We show that TIEG1/KLF10 is transcriptionally activated by E2F1 and crucial for E2F1-mediated chemosensitization of cancer cells. Our results provide a broader picture of E2F1-regulated genes in conjunction with cytotoxic treatment that allows the design of more rational therapeutics.

Deletion of the p107/p130-binding domain of Mip130/LIN-9 bypasses the requirement for CDK4 activity for the dissociation of Mip130/LIN-9 from p107/p130-E2F4 complex

Mip130/LIN-9 is part of a large complex that includes homologs of the Drosophila dREAM (drosophila RB-like, E2F, and Myb) and C. elegans DRM complexes. This complex also includes proteins such as Mip40/LIN-37, Mip120/LIN-54, and LIN-52. In mammalian cells, Mip130/LIN-9 specifically associates with the p107/p130-E2F4 repressor complex in G0/G1 and with B-Myb in S-phase. However, little is known about how the transition occurs and whether Mip130/LIN-9 contributes to the repressor effect of p107/p130. In this report, we demonstrate that Mip130/LIN-9, Mip40/LIN-37, Mip120/LIN-54, and Sin3b form a core complex, the Mip Core Complex or LIN Complex (MCC/LINC), which is detectable in all phases of the cell cycle. This complex specifically recruits transcriptional repressors such as p107, p130, E2F4 and HDAC1 in G0/G1, and B-Myb in S-phase. Importantly, we provide strong evidence that the transition between repressors and activators of transcription is mediated by CDK4, through the phosphorylation of the pocket proteins, p107 and p130. The requirement for CDK4 activity is bypassed by the deletion of the first 84 amino acids (Mip130/LIN-9Δ84), since this mutant is unable to interact with p107/p130 in G0/G1, while maintaining its association with B-Myb. Importantly, the Mip130/LIN-9Δ84 allele rescues the low expression of G1/S genes observed in CDK4−/− MEFs demonstrating that Mip130/LIN-9 contributes to the repression of these E2F-regulated genes in G0/G1.

Modulation of the E2F1-Driven Cancer Cell Fate by the DNA Damage Response Machinery and Potential Novel E2F1 Targets in Osteosarcomas

Osteosarcoma is the most common primary bone cancer. Mutations of the RB gene represent the most frequent molecular defect in this malignancy. A major consequence of this alteration is that the activity of the key cell cycle regulator E2F1 is unleashed from the inhibitory effects of pRb. Studies in animal models and in human cancers have shown that deregulated E2F1 overexpression possesses either "oncogenic" or "oncosuppressor" properties, depending on the cellular context. To address this issue in osteosarcomas, we examined the status of E2F1 relative to cell proliferation and apoptosis in a clinical setting of human primary osteosarcomas and in E2F1-inducible osteosarcoma cell line models that are wild-type and deficient for p53. Collectively, our data demonstrated that high E2F1 levels exerted a growth-suppressing effect that relied on the integrity of the DNA damage response network. Surprisingly, induction of p73, an established E2F1 target, was also DNA damage response-dependent. Furthermore, a global proteome analysis associated with bioinformatics revealed novel E2F1-regulated genes and potential E2F1-driven signaling networks that could provide useful targets in challenging this aggressive neoplasm by innovative therapies.

Mammalian target of rapamycin inhibitors rapamycin and RAD001 (everolimus) induce anti-proliferative effects in GH-secreting pituitary tumor cells in vitro

The effect of mammalian target of rapamycin (mTOR) inhibitors on pituitary tumors is unknown. Akt overexpression was demonstrated in pituitary adenomas, which may render them sensitive to the anti-proliferative effects of these drugs. The objective of the study was to evaluate the anti-proliferative efficacy of the mTOR inhibitor, rapamycin, and its orally bioavailable analog RAD001 on the GH-secreting pituitary tumor GH3 and MtT/S cells and in human GH-secreting pituitary adenomas (GH-omas) in primary cell cultures. Treatment with rapamycin or RAD001 significantly decreased the number of viable cells and cell proliferation in a dose- and time-dependent manner. This was reflected by decreased phosphorylation levels of the downstream mTOR target p70S6K. Rapamycin treatment of GH3 cells induced G0/G1 cell cycle arrest. In other tumor cell types, this was attributed to a decrease in cyclin D1 levels. However, rapamycin did not affect cyclin D1 protein levels in GH3 cells. By contrast, it decreased cyclin D3 and p21/CIP, which stabilizes cyclin D/cyclin-dependent kinase 4 (cdk4) complexes. Rapamycin inhibited FCS-induced retinoblastoma phosphorylation and subsequent E2F-transcriptional activity. In response to decreased E2F activity, the expression of the E2F-regulated genes cyclin E and cdk2 was reduced. Our results showed that mTOR inhibitors potently inhibit pituitary cell proliferation, suggesting that mTOR inhibition may be a promising anti-proliferative therapy for pituitary adenomas. This therapeutic manipulation may have beneficial effects particularly for patients harboring invasive pituitary tumors resistant to current treatments.

Quantitative RNA expression analysis with Affymetrix Tiling 1.0R arrays identifies new E2F target genes

The Affymetrix ATH1 array provides a robust standard tool for transcriptome analysis, but unfortunately does not represent all of the transcribed genes in Arabidopsis thaliana. Recently, Affymetrix has introduced its Arabidopsis Tiling 1.0R array, which offers whole-genome coverage of the sequenced Col-0 reference strain. Here, we present an approach to exploit this platform for quantitative mRNA expression analysis, and compare the results with those obtained using ATH1 arrays. We also propose a method for selecting unique tiling probes for each annotated gene or transcript in the most current genome annotation, TAIR7, generating Chip Definition Files for the Tiling 1.0R array. As a test case, we compared the transcriptome of wild-type plants with that of transgenic plants overproducing the heterodimeric E2Fa-DPa transcription factor. We show that with the appropriate data pre-processing, the estimated changes per gene for those with significantly different expression levels is very similar for the two array types. With the tiling arrays we could identify 368 new E2F-regulated genes, with a large fraction including an E2F motif in the promoter. The latter groups increase the number of excellent candidates for new, direct E2F targets by almost twofold, from 181 to 334.

Gene expression reveals two distinct groups of anal carcinomas with clinical implications

Human papillomavirus (HPV) is a major aetiological agent in anal carcinomas. We here present a study of global gene expression using microarray hybridisation in a collection of anal carcinoma biopsies. Quantitative PCR was used to verify expression of selected genes. All biopsies contained integrated DNA of human papillomavirus subtype 16 (HPV16) and expressed HPV16 E7 mRNA. No other subspecies of HPV were detected in these 13 biopsies as assessed by PCR amplification and DNA sequencing. Unsupervised cluster analysis, based on global mRNA expression, divided the tumour biopsies into two distinct groups. Cluster analysis based on a number of high-risk HPV and/or E2F-regulated genes reproduced this biopsy grouping, suggesting that integrated HPV16 substantially influenced global gene expression in approximately half the biopsies studied. The levels of HPV16 E7 mRNA were significantly different between the two groups, but with considerable overlap. Thus, influence on global gene expression could not be absolutely ascribed to the expression level of HPV16. To investigate whether this distinction in gene expression had prognostic impact, we studied protein expression in an independent cohort of 55 anal carcinomas not included in the microarray study of two differentially expressed candidate genes, minichromosome maintenance complex component 7 (MCM7) and cyclin-dependent kinase inhibitor 2A (CDKN2A or p16). HPV status was assessed by in situ hybridisation. There was a significant association between in situ staining for HPV E7 mRNA and immunostaining for CDKN2A (p16) and MCM7 protein. CDKN2A (p16) mRNA was found significantly differentially expressed between the two tumour groups. However, cluster analysis on genes directly regulated by CDKN2A (p16) could not reproduce this split of biopsies into two groups, suggesting that the transcriptional regulatory activity of CDKN2A in these biopsies is inhibited. Furthermore, protein expression of CDKN2A (p16) could not be associated with survival. MCM7 is directly regulated by E2F and induced by HPV, and its mRNA was found differentially expressed between the two tumour groups. High level of MCM7 protein was found to be associated with both improved relapse-free survival (RFS, P=0.02) and cancer-specific survival (CSS, P=0.03) in anal cancer patients treated with radiation with or without additional chemotherapy.

S-Phase-specific Activation of PKCα Induces Senescence in Non-small Cell Lung Cancer Cells

Protein kinase C (PKC) has been widely implicated in positive and negative control of cell proliferation. We have recently shown that treatment of non-small cell lung cancer (NSCLC) cells with phorbol 12-myristate 13-acetate (PMA) during G1 phase inhibits the progression into S phase, an effect mediated by PKC delta-induced up-regulation of the cell cycle inhibitor p21 Cip1. However, PMA treatment in asynchronously growing NSCLC cells leads to accumulation of cells in G2/M. Studies in post-G1 phases revealed that PMA induced an irreversible G2/M cell cycle arrest in NSCLC cells and conferred morphological and biochemical features of senescence, including elevated SA-beta-Gal activity and reduced telomerase activity. Remarkably, this effect was phase-specific, as it occurred only when PKC was activated in S, but not in G1, phase. Mechanistic analysis revealed a crucial role for the classical PKC alpha isozyme as mediator of the G2/M arrest and senescence, as well as for inducing p21(Cip1) an obligatory event for conferring the senescence phenotype. In addition to the unappreciated role of PKC isozymes, and specifically PKC alpha, in senescence, our data introduce the paradigm that discrete PKCs trigger distinctive responses when activated in different phases of the cell cycle via a common mechanism that involves p21 Cip1 up-regulation.

Arabidopsis RETINOBLASTOMA-RELATED PROTEIN 1 is involved in G1 phase cell cycle arrest caused by sucrose starvation

Although sucrose availability is crucial for commitment to plant cell division during G1 phase by controlling the expression of D-type cyclins, it has remained unclear how these factors mediate entry into the cell cycle. Here we show that Arabidopsis RETINOBLASTOMA-RELATED PROTEIN 1 (AtRBR1) is involved in G1-phase cell cycle arrest caused by sucrose starvation. We generated estrogen-inducible AtRBR1 RNA interference (RNAi) Arabidopsis suspension MM2d cells, and found that downregulation of AtRBR1 leads to a higher frequency of arrest in G2 phase, instead of G1-phase arrest in the uninduced control, after sucrose starvation. Synchronization experiments confirmed that downregulation of AtRBR1 leads to a prolonged G2 phase and delayed activation of G2/M marker genes. Downregulation of AtRBR1 also stimulated the activation of E2F-regulated genes when these genes were repressed in the uninduced cells under the limited sucrose conditions. We conclude that AtRBR1 is a key effector for the ability of sucrose to modulate progression from G1 phase.

Cyclin D1/cdk4 can interact with E2F4/DP1 and disrupts its DNA‐binding capacity

The E2F family of transcription factors regulate the expression of many growth-related genes in a cell cycle-dependent manner. These transcription factors can activate or, in conjunction with an Rb-related protein, repress transcription. E2F transcriptional activity is regulated at several different levels that are each linked to cell cycle progression. In many cell types, E2F4 and E2F5 are the predominant E2F species during G(0) and early G(1) and function primarily as repressors of E2F-regulated genes. In this study, co-immunoprecipitation techniques were used to demonstrate that cyclins D1, D2, and D3 are capable of interacting with E2F4, E2F5, and DP1. Overexpression of cyclin D1/cdk4 reduced E2F4-mediated transcription in a simple reporter gene assay and electrophoretic mobility shift analyses using nuclear extracts from transfected cells indicated that cyclin D1/cdk4 disrupts the DNA-binding ability of E2F4. Cell cycle analysis following stimulation of serum-starved 3T3 cells indicated that E2F4 undergoes changes in its phosphorylation pattern coincident with the synthesis of cyclin D1. Examination of a series of E2F4 deletion mutants indicated that a cyclin D1-binding site located close to the carboxyl terminus of E2F4 was critical for the disruption of DNA binding by cyclin D1/cdk4. These data support a model in which E2F4 DNA binding is abolished during mid-G(1) at the same time when E2F interactions with pRb-related proteins are disrupted by cyclin D1/cdk4.

C/EBPβ Activates E2F-regulated Genes in Vivo via Recruitment of the Coactivator CREB-binding Protein/P300

The E2F transcription factors play an essential role in regulating the G(1)- to S-phase transition of the cell cycle. Previous studies have identified the importance of interactions between E2Fs and other transcription factors as a mechanism for transcriptional control of a subset of E2F regulated target genes. However, the mechanisms responsible for E2F target gene specificity remain incompletely understood. Here we report that in a mammalian in vivo model of synchronized proliferation, C/EBPbeta occupancy on the promoters of E2F-regulated growth-related genes increases as a function of cell cycle progression. C/EPBbeta binding to these promoters is associated with recruitment of the coactivator CBP/p300, histone H4 acetylation, and maximal activation of E2F target genes. Moreover, binding of CBP/p300 to E2F targets is markedly reduced in C/EBPbeta null mice, resulting in reduced expression of E2F regulated genes. These findings identify C/EBPbeta as a direct activator of E2F target genes in mammalian cell cycle progression through a mechanism that involves recruitment of CBP/p300. The demonstration of a functional link between C/EBPbeta and CBP/p300 for E2F target gene activation provides a potential mechanism for how coactivators such as CBP/p300 can be selectively recruited to E2F target genes in response to tissue-specific growth stimuli.

The AP-1 transcription factor regulates breast cancer cell growth via cyclins and E2F factors

The activating protein-1 (AP-1) transcription factor transduces growth signals through signal transduction pathways to the nucleus, leading to the expression of genes involved in growth and malignant transformation in many cell types. We have previously shown that overexpression of a dominant negative form of the cJun proto-oncogene, a cJun dominant negative mutant (Tam67), blocks AP-1 transcriptional activity, induces a G(1) cell cycle block and inhibits breast cancer cell growth in vitro and in vivo. We found that AP-1 blockade by Tam67 in MCF-7 breast cancer cells downregulates cyclin D1 transcriptional activity by at least two mechanisms: by suppressing transcription at the known AP-1 binding site (-934/-928) and by suppressing growth factor-induced expression through suppressing E2F activation at the E2F-responsive site (-726/-719). AP-1 blockade also led to reduced expression of E2F1 and E2F2, but not E2F4, at the mRNA and protein levels. Chromatin immunoprecipitation and supershift assays demonstrated that AP-1 blockade caused decreased binding of E2F1 protein to the E2F site in the cyclin D1 promoter. We also found that Tam67 suppressed the expression of the E2F1 dimerizing partner, DP1 and E2F-upregulated cell cycle genes (cyclins E, A, B and D3) and enhanced the expression of E2F-downregulated cell cycle genes (cyclins G(2) and I). Reduced expression of other E2F-regulated genes was also seen with AP-1 blockade and E2F suppression. Thus, the AP-1 factor regulates the expression of cyclin D and E2F (the latter in turn regulates E2F-downstream genes), leading to cell cycle progression and breast cancer cell proliferation.

Setting the Stage for S Phase

In a recent issue of Molecular Cell, Tyagi et al. (2007) show that E2F1, a positive regulator of S phase entry, recruits cofactor HCF-1 and associated hSet1/MLL histone H3 lysine 4 methyltransferase complex, facilitating the activation of genes required for proliferation.

Mifepristone Inhibits Ovarian Cancer Cell GrowthIn vitroandIn vivo

These studies were designed to determine whether the synthetic steroid mifepristone inhibits ovarian cancer growth in vitro and in vivo and the molecular mechanisms involved. The effect of mifepristone on ovarian cancer cell growth in vitro was studied in ovarian cancer cell lines of different genetic backgrounds (SK-OV-3, Caov-3, OV2008, and IGROV-1). In addition, the growth inhibition capacity of mifepristone on ovarian carcinoma xenografts was tested in nude mice. Mifepristone inhibited ovarian cancer cell proliferation in a dose- and time-dependent manner. The cytostatic effect of mifepristone was confirmed in a clonogenic survival assay and was not linked to loss of viability. Mifepristone blocked DNA synthesis, arrested the cell cycle at the G(1)-S transition, up-regulated cyclin-dependent kinase (cdk) inhibitors p21(cip1)and p27(kip1), down-regulated transcription factor E2F1, decreased expression of the E2F1-regulated genes cdk1 (cdc2) and cyclin A, and modestly decreased cdk2 and cyclin E levels. The abrupt arrest in cell growth induced by mifepristone correlated with reduced cdk2 activity, increased association of cdk2 with p21(cip1) and p27(kip1), increased nuclear localization of the cdk inhibitors, and reduced nuclear abundance of cdk2 and cyclin E. In vivo, mifepristone significantly delayed the growth of ovarian carcinoma xenografts in a dose-dependent manner and without apparent toxic effects for the animals. These preclinical studies show that mifepristone is effective as a single agent in vitro and in vivo, inhibiting the growth of human epithelial ovarian cancer cells. Mifepristone markedly reduces cdk2 activity likely due to increased association of cdk2 with the cdk inhibitors p21(cip1) and p27(kip1) and reduced nuclear cdk2/cyclin E complex availability. Acting as a cytostatic agent, mifepristone promises to be of translational significance in ovarian cancer therapeutics.

Apoptotic action of E2F1 requires glycogen synthase kinase 3‐β activity in PC12 cells

Both E2F1 and GSK3beta have been described as essential targets in neuronal apoptosis. Previous studies have demonstrated that GSK3beta binds to E2F1 in vivo. We wanted to investigate whether these proteins could share a common apoptotic signal pathway in neuronal cells. With this intention, we developed a PC12 ER-E2F1 stable cell line in which E2F1 activity was dependent on the presence of 4-hydroxitamoxifen. E2F1 activation produced apoptosis in naive and post-mitotic cells; serum and nerve growth factor respectively protected them from E2F1 apoptotic stimuli. The presence of specific GSK3beta inhibitors SB216763 and LiCl completely protected cells from apoptosis induced by E2F1 activation. In addition, knocked down GSK3beta experiments by small interference RNAs have demonstrated that a reduction of GSK3beta protein levels can lower the apoptotic effect of E2F1. Finally, we demonstrated that the apoptotic effect of E2F1 is not due to the regulation of GSK3beta activity, and that the inhibitory effect of GSK3beta inhibitor SB216763 on E2F1 induced apoptosis could be due to an alteration in the E2F1-regulated transcription gene pattern. In summary, we have demonstrated that the apoptotic action of E2F1 requires GSK3beta activity.

Transcriptome profiling of the C. elegans Rb ortholog reveals diverse developmental roles

LIN-35 is the single C. elegans ortholog of the mammalian pocket protein family members, pRb, p107, and p130. To gain insight into the roles of pocket proteins during development, a microarray analysis was performed with lin-35 mutants. Stage-specific regulation patterns were revealed, indicating that LIN-35 plays diverse roles at distinct developmental stages. LIN-35 was found to repress the expression of many genes involved in cell proliferation in larvae, an activity that is carried out in conjunction with E2F. In addition, LIN-35 was found to regulate neuronal genes during embryogenesis and targets of the intestinal-specific GATA transcription factor, ELT-2, at multiple developmental stages. Additional findings suggest that LIN-35 functions in cell cycle regulation in embryos in a manner that is independent of E2F. A comparison of LIN-35-regulated genes with known fly and mammalian pocket protein targets revealed a high degree of overlap, indicating strong conservation of pocket protein functions in diverse phyla. Based on microarray results and our refinement of the C. elegans E2F consensus sequence, we were able to generate a comprehensive list of putative E2F-regulated genes in C. elegans. These results implicate a large number of genes previously unconnected to cell cycle control as having potential roles in this process.

The retinoblastoma tumor suppressor modifies the therapeutic response of breast cancer

The retinoblastoma tumor suppressor (RB) protein is functionally inactivated in the majority of human cancers and is aberrant in one-third of all breast cancers. RB regulates G(1)/S-phase cell-cycle progression and is a critical mediator of antiproliferative signaling. Here the specific impact of RB deficiency on E2F-regulated gene expression, tumorigenic proliferation, and the response to 2 distinct lines of therapy was investigated in breast cancer cells. RB knockdown resulted in RB/E2F target gene deregulation and accelerated tumorigenic proliferation, thereby demonstrating that even in the context of a complex tumor cell genome, RB status exerts significant control over proliferation. Furthermore, the RB deficiency compromised the short-term cell-cycle inhibition following cisplatin, ionizing radiation, and antiestrogen therapy. In the context of DNA-damaging agents, this bypass resulted in increased sensitivity to these agents in cell culture and xenograft models. In contrast, the bypass of antiestrogen signaling resulted in continued proliferation and xenograft tumor growth in the presence of tamoxifen. These effects of aberrations in RB function were recapitulated by ectopic E2F expression, indicating that control of downstream target genes was an important determinant of the observed responses. Specific analyses of an RB gene expression signature in 60 human patients indicated that deregulation of this pathway was associated with early recurrence following tamoxifen monotherapy. Thus, because the RB pathway is a critical determinant of tumorigenic proliferation and differential therapeutic response, it may represent a critical basis for directing therapy in the treatment of breast cancer.