E2F Family Of Transcription Factors Research Articles

Abstract 2344: Beyond RB1: the pocket protein p130 mediates the effects of CDK4/6 inhibition in RB1-deficient urothelial carcinoma

Abstract Introduction: Urothelial carcinoma is characterized by a high incidence of molecular alterations in the CDKN2A-RB-E2F axis. Loss of the cyclin-dependent kinase inhibitor p16 encoded by CDKN2A or amplification of CDK4/6 is associated with sensitivity to CDK4/6 inhibitors in various cancers. Functional RB1 is believed to be canonically required for mediating the effects of CDK4/6 inhibition in this setting. However, muscle-invasive urothelial carcinomas harbor RB mutations and copy-number losses in 21% of patients. To extend the activity spectrum of CDK4/6 inhibitors to these RB1-deficient cancers, we investigated the role of pocket protein p130 encoded by the Retinoblastoma-Like 2 (RBL2) gene, in mediating the downstream effects of CDK4/6 inhibition. Methods: CRISPR-Cas9 was used for CDKN2A knockout in the 5637-urothelial cancer cell line with an RB-mutant background and in TCCSUP cell line with wild-type RB. CDKN2A- cells were subsequently treated with CDK4/6 inhibitor palbociclib. Levels of RB1, phospho-RB1 (at serine 807/ serine 811), p130 and phospho-p130 (specifically, the CDK4/6-dependent serine-672 phosphorylation) proteins were quantified using SDS-PAGE. Cell-cycle phase analysis was performed using flow cytometric measurement of BrdU and PI staining. Results: CRISPR-induced disruption of CDKN2A was validated using genomic PCR and targeted Miseq sequencing. p16 protein knockdown was validated using SDS-PAGE. CRISPR-induced CDKN2A knockdown resulted in increased cell proliferation and faster G1/S transition even in the absence of functional RB in the 5637-urothelial cancer cell line. A significant increase in the levels of phospho-p130 was observed after p16 knockdown without changes in the levels of unphosphorylated p130. TCCSUP cell line with functional RB demonstrated a similar increase in proliferation, faster G1/S transition and an increase in phospho-p130. Palbociclib was effective in reversing these changes in all the tested cell lines. Conclusions: the pocket protein p130 plays an important role in mediating downstream effects of CDK4/6 inhibition in urothelial cancer cell lines with both wild-type and more importantly, deficient RB. CDK4/6-dependent phosphorylation of p130 can effectively compensate for the functional loss of RB activating E2F family of transcription factors that control G1/S checkpoint. These findings potentially explain the lack of correlation between RB and response to CDK4/6 inhibitors in clinical trials. These results provide a mechanistic rationale for extending clinical trials of CDK4/6 inhibitors to patients with RB1-deficient tumors. Citation Format: Bishoy M. Faltas, Ethan Shelkey, Rebecca Meyer, Nathan Young, Mark A. Rubin. Beyond RB1: the pocket protein p130 mediates the effects of CDK4/6 inhibition in RB1-deficient urothelial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2344. doi:10.1158/1538-7445.AM2017-2344

Abstract 488: E2F2-mediated copy number changes drive metastasis and therapeutic response of HER2-positive tumors through Col1a1, CHAD, and AKT-dependent mechanisms

Abstract The E2F family of transcription factors is classically known to regulate G1 to S-phase transition in cell cycle but has emerging roles in HER2+ breast cancer. A loss of E2F1 or E2F2, in a HER2 mouse model, MMTV-Neu, leads to a decrease in tumor metastasis1. It is not known what mechanistic roles specific E2Fs are playing in this process. To investigate this, we leveraged and bioinformatic principles, including genomics and transcriptomics with traditional laboratory science and high throughput drug screening projects. This experimental approach immediately revealed that loss of E2F2 is significantly associated with a more unstable tumor including an increase in copy number alterations, single nucleotide variants, and translocations. Further analysis revealed a conserved copy number alteration in both mouse and humans. Specifically we noted the amplification of 17q21.33 in 25% of HER2+ patients. The analogous region chromosome 11D was lost in 30% of the less metastatic MMTV-Neu E2F2 knockout mice indicating a role of the region in tumor metastasis. Transcriptomic data revealed that two genes, Collagen Type I, alpha 1 (Col1a1) and Chondroadherin (CHAD), were potential genes of significance in the amplification event. CRISPR mediated knockout studies were conducted to determine each gene’s effect on tumor cell migration and metastasis in mouse and human derived cell lines. Wound healing assays and tail vein injection have shown the Col1a1 and CHAD KO cell lines have a delay in cell migration (P<.01) and reduced ability to colonize the lung (P<.05) respectively. Oncogenic signaling data shows that the 17q21.33 amplification event has higher AKT and E2F2 signaling than HER2 positive tumors without the event. It was hypothesized that the tumors would be dependent on the signaling and perturbation of the network might be an effective therapy for patients with the 17q21.33 amplification event. To investigate this we identified deferentially lethal siRNAs and compounds between HER2+ tumors with and without the 17q21.33 event in the Achilles, CCLE, and PDX datasets. A String-DB analysis showed many of the deferentially lethal genes and compounds centered around AKT. A decrease in AKT signaling through siRNA or chemical compound results in the death of the cell in the 17q21.33 amplified samples but not the HER2+ samples without the event. This study reveals that patients’ with a 17q21.33 amplification event have more metastatic tumors mediated through Col1a1 and CHAD and may be responsive to AKT targeting therapy.

TGF-β induces growth suppression in multiple myeloma MM.1S cells via E2F1.

Transforming growth factor-β (TGF-β) has an important role in multiple target genes and signaling pathways. The E2F family of transcription factors is a group of DNA-binding proteins that are involved in cell-cycle progression, and therefore have a key role in proliferation. The present study demonstrates that inhibition of cell growth by TGF-β occurs in the multiple myeloma cell line MM.1S. However, the growth-suppressive effects of TGF-β may be reversed by small interfering (si)RNA to reduce the expression of E2F1. TGF-β1 and E2F1 siRNA were manipulated in MM.1S cells to investigate the association between these genes. FACScan Flow Cytometer, western blot analysis and other methods were adopted to confirm such interrelation. The present data showed that TGF-β mediated growth suppression in MM.1S cells, while inducing E2F1 protein expression levels rapidly and transiently. The present data support the hypothesis that E2F1 is a central mediator of TGF-β-induced growth suppression in MM.1S cells and control of E2F1 may be a downstream event of TGF-β action, at least in one multiple myeloma cell line.

Molecular cloning, expression, and characterization of E2F transcription factor 4 from Antheraea pernyi.

The E2F transcription factor family is distributed widely in eukaryotes and has been well studied among mammals. In the present study, the E2F transcription factor 4 (E2F4) gene was isolated from fat bodies of Antheraea pernyi and sequenced. E2F4 comprised a 795 bp open reading frame encoding a deduced amino acid sequence of 264 amino acid residues. The recombinant protein was expressed in Escherichia coli (Transetta DE3), and anti-E2F4 antibodies were prepared. The deduced amino acid sequence displayed significant homology to an E2F4-like protein from Bombyx mori L. Quantitative real-time polymerase chain reaction analysis revealed that E2F4 expression was highest in the integument, followed by the fat body, silk glands, and haemocytes. The expression of E2F4 was upregulated in larvae challenged by bacterial (Escherichia coli, Micrococcus luteus), viral (nuclear polyhedrosis virus), and fungal (Beauveria bassiana) pathogens. These observations indicated that E2F4 is an inducible protein in the immune response of A. pernyi and probably in other insects.

Conservation and divergence of C-terminal domain structure in the retinoblastoma protein family

The retinoblastoma protein (Rb) and the homologous pocket proteins p107 and p130 negatively regulate cell proliferation by binding and inhibiting members of the E2F transcription factor family. The structural features that distinguish Rb from other pocket proteins have been unclear but are critical for understanding their functional diversity and determining why Rb has unique tumor suppressor activities. We describe here important differences in how the Rb and p107 C-terminal domains (CTDs) associate with the coiled-coil and marked-box domains (CMs) of E2Fs. We find that although CTD-CM binding is conserved across protein families, Rb and p107 CTDs show clear preferences for different E2Fs. A crystal structure of the p107 CTD bound to E2F5 and its dimer partner DP1 reveals the molecular basis for pocket protein-E2F binding specificity and how cyclin-dependent kinases differentially regulate pocket proteins through CTD phosphorylation. Our structural and biochemical data together with phylogenetic analyses of Rb and E2F proteins support the conclusion that Rb evolved specific structural motifs that confer its unique capacity to bind with high affinity those E2Fs that are the most potent activators of the cell cycle.

Abstract A11: RB localizes to DNA double strand breaks and promotes DNA end resection and homologous recombination through the recruitment of BRG1

Abstract The retinoblastoma (RB) tumor suppressor is widely recognized as a master regulator of the transcriptional program that controls entry into the S phase of the cell cycle. Its loss, which is a hallmark of cancer, leads to uncontrolled cell proliferation. RB works by binding to members of the E2F family of transcription factors and recruiting chromatin modifiers and remodelers to the promoters of E2F target genes. Here we show that RB also localizes to DNA double strand breaks (DSBs) dependent on E2F1 and ATM kinase activity. RB promotes DNA end resection and DSB repair through homologous recombination (HR), and its loss results in genome instability. RB is necessary for the recruitment of the BRG1 ATPase to DSBs, which stimulates DNA end resection and HR. A knock-in mutation of the ATM phosphorylation site on E2F1 (S29A) prevents the interaction between E2F1 and TopBP1 and recruitment of RB, E2F1 and BRG1 to DSBs. This knock-in mutation also impairs DNA repair, increases genomic instability and renders mice hypersensitive to IR. Importantly, depletion of either RB or BRG1 in osteosarcoma and breast cancer cell lines results in sensitivity to DNA damaging agents, thus underscoring the importance of these factors in DNA repair. We uncover a novel, non-transcriptional function for RB in HR, which could contribute to genome instability associated with RB loss. Citation Format: Renier Vélez-Cruz, Swarnalatha Manickavinayaham, Anup K. Biswas, Regina W. Clary, David G. Johnson. RB localizes to DNA double strand breaks and promotes DNA end resection and homologous recombination through the recruitment of BRG1 [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A11.

Dosage-dependent copy number gains in E2f1 and E2f3 drive hepatocellular carcinoma.

Disruption of the retinoblastoma (RB) tumor suppressor pathway, either through genetic mutation of upstream regulatory components or mutation of RB1 itself, is believed to be a required event in cancer. However, genetic alterations in the RB-regulated E2F family of transcription factors are infrequent, casting doubt on a direct role for E2Fs in driving cancer. In this work, a mutation analysis of human cancer revealed subtle but impactful copy number gains in E2F1 and E2F3 in hepatocellular carcinoma (HCC). Using a series of loss- and gain-of-function alleles to dial E2F transcriptional output, we have shown that copy number gains in E2f1 or E2f3b resulted in dosage-dependent spontaneous HCC in mice without the involvement of additional organs. Conversely, germ-line loss of E2f1 or E2f3b, but not E2f3a, protected mice against HCC. Combinatorial mapping of chromatin occupancy and transcriptome profiling identified an E2F1- and E2F3B-driven transcriptional program that was associated with development and progression of HCC. These findings demonstrate a direct and cell-autonomous role for E2F activators in human cancer.

Identification of a Low-Frequency Missense Variant in E2F Transcription Factor 7 Associated with Colorectal Cancer Risk In A Chinese Population

Background:Transcription factors regulate gene expression and play important role in tumor genesis. Especially, the E2F transcription factor family controls the cell cycle and regulate many tumor suppressors. Missense variants in E2F family genes, which change the amino acid sequence, may alter the capacity for DNA binding or the protein structure, leading to a functional alteration.Material and Methods:We here searched for missense variants in E2F transcription family genes (E2F1~E2F8) and identified two (rs2075995 for E2F2 and rs3829295 for E2F7) with minor allele frequencies >0.01 in Chinese Han Beijing population from the 1000 genome project. We genotyped these two variants in 1,055 colorectal cancer (CRC) patients and 1,936 healthy controls using Taqman genotyping assays and assessed associations between SNPs and risk of CRC using logistic regression adjusted for gender and age.Results:We found rs3829295 at E2F7 to be significantly associated with risk of CRC. Compared with TT genotype carriers, CT and CT+CC genotype carriers had lower risks of CRC with ORs of 0.61 (95% CI: 0.44-0.85, P=0.003) and 0.61 (95% CI: 0.44-0.84, P=0.003), respectively. When stratified by gender and age, significant associations were observed in males (OR= 0.56, 95% CI: 0.38-0.83, P=0.004) for rs3829295, but not females (OR= 0.73, 95% CI: 0.43-1.22, P=0.232).Conclusion:Through a systematic assessment of variants in the E2F transcription factor family, we identified a low-frequent missense variant in E2F7 significantly associated with CRC risk, indicating that E2F7 may play an important role in development of this tumor type.

Progress with palbociclib in breast cancer: latest evidence and clinical considerations.

Deregulation of the cell cycle is a hallmark of cancer, and research on cell cycle control has allowed identification of potential targets for anticancer treatment. Palbociclib is a selective inhibitor of the cyclin-dependent kinases 4 and 6 (CDK4/6), which are involved, with their coregulatory partners cyclin D, in the G1-S transition. Inhibition of this step halts cell cycle progression in cells in which the involved pathway, including the retinoblastoma protein (Rb) and the E2F family of transcription factors, is functioning, although having been deregulated. Among breast cancers, those with functioning cyclin D-CDK4/6-Rb-E2F are mainly hormone-receptor (HR) positive, with some HER2-positive and rare triple-negative cases. Deregulation results from genetic or otherwise occurring hyperactivation of molecules subtending cell cycle progression, or inactivation of cell cycle inhibitors. Based on results of randomized clinical trials, palbociclib was granted accelerated approval by the US Food and Drug Administration (FDA) for use in combination with letrozole as initial endocrine-based therapy for metastatic disease in postmenopausal women with HR-positive, HER2-negative breast cancer, and was approved for use in combination with fulvestrant in women with HR-positive, HER2-negative advanced breast cancer with disease progression following endocrine therapy. This review provides an update of the available knowledge on the cell cycle and its regulation, on the alterations in cyclin D-CDK4/6-Rb-E2F axis in breast cancer and their roles in endocrine resistance, on the preclinical activity of CDK4/6 inhibitors in breast cancer, both as monotherapy and as partners of combinatorial synergic treatments, and on the clinical development of palbociclib in breast cancer.

RB localizes to DNA double-strand breaks and promotes DNA end resection and homologous recombination through the recruitment of BRG1.

The retinoblastoma (RB) tumor suppressor is recognized as a master regulator that controls entry into the S phase of the cell cycle. Its loss leads to uncontrolled cell proliferation and is a hallmark of cancer. RB works by binding to members of the E2F family of transcription factors and recruiting chromatin modifiers to the promoters of E2F target genes. Here we show that RB also localizes to DNA double-strand breaks (DSBs) dependent on E2F1 and ATM kinase activity and promotes DSB repair through homologous recombination (HR), and its loss results in genome instability. RB is necessary for the recruitment of the BRG1 ATPase to DSBs, which stimulates DNA end resection and HR. A knock-in mutation of the ATM phosphorylation site on E2F1 (S29A) prevents the interaction between E2F1 and TopBP1 and recruitment of RB, E2F1, and BRG1 to DSBs. This knock-in mutation also impairs DNA repair, increases genomic instability, and renders mice hypersensitive to IR. Importantly, depletion of RB in osteosarcoma and breast cancer cell lines results in sensitivity to DNA-damaging drugs, which is further exacerbated by poly-ADP ribose polymerase (PARP) inhibitors. We uncovered a novel, nontranscriptional function for RB in HR, which could contribute to genome instability associated with RB loss.

Abstract B11: Re-wired E2F function in response to RB loss as a potential driver of castration-resistant prostate cancer

Abstract Prostate cancer (PCa) is the most frequently diagnosed non-cutaneous malignancy, and second leading cause of cancer death in males in the United States, causing over 30,000 deaths per year. A crucial component to the development and progression of PCa is the activity of the androgen receptor (AR). AR promotes proliferation and is required for PCa cell growth and survival. As such, targeting the AR-signaling axis through androgen deprivation therapy (ADT) is the first line of therapy for PCa. However, cells invariably become resistant to this therapy and men relapse with the incurable form of the disease, castration-resistant prostate cancer (CRPC). It has been shown that an underlying mechanism of this resistance revolves around the retinoblastoma (RB) tumor suppressor protein. RB functions to repress tumor development by negatively regulating the activity of the E2F family of transcription factors thus, resulting in a decrease in AR levels. RB additionally controls cell cycle progression through its ability to bind and inhibit E2F1 mediated transcription of genes required for G1/S progression. Surprisingly, genome-wide assessment of E2F activity in the absence of RB has been understudied. To better understand the molecular pathways that are altered upon RB loss and the roles they play in CRPC, RNA-Seq and ChIP-Seq analyses were performed in isogenic RB cell lines in both hormone sensitive and CRPC models. Data to be discussed reveal new insight into the differential effects of RB loss on AR and E2F1 function and role as potential drivers of late stage disease. Citation Format: Amy C. Mandigo, Chris McNair, Matthew J. Schiewer, Kexin Xu, Fugen Li, Brown Myles, Karen E. Knudsen. Re-wired E2F function in response to RB loss as a potential driver of castration-resistant prostate cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr B11.

Abstract PR05: RB localizes to DNA double strand breaks and promotes DNA end resection and homologous recombination through the recruitment of SWI/SNF complex

Abstract The retinoblastoma (RB) tumor suppressor is widely recognized as a master regulator of the transcriptional program that controls entry into the S phase of the cell cycle. RB works by binding to members of the E2F family of transcription factors and recruiting chromatin-remodelers and modifiers to the promoters of E2F target genes. Here we show that RB localizes to DNA double strand breaks (DSBs) dependent on E2F1 and ATM kinase activity. Moreover, RB promotes DNA end resection and homologous recombination, and its loss results in genome instability. RB mediates this important role in repair by recruiting the SWI/SNF chromatin remodeler BRG1 to DSBs. In agreement with these results, loss of BRG1 also results in impaired DNA end resection and HR. RB is recruited to DSBs through a DNA damage-inducible phospho-specific interaction between E2F1 and TopBP1. A knock-in mutation of the ATM phosphorylation site on E2F1 (S29A) prevents the interaction between E2F1 and TopBP1 and recruitment of RB, E2F1 and BRG1 to DSBs. This E2F1 mutation also impairs DNA repair, increases genomic instability and renders knock-in mice hypersensitive to IR. We uncover a novel, non-transcriptional function for RB in HR, which could contribute to genome instability associated with RB loss in human cancers and explain their sensitivity to DSB-inducing agents. This abstract is also being presented as Poster B09. Citation Format: Renier Velez-Cruz, Swarnalatha Manickavinayaham, Anup K. Biswas, Regina Weaks Clary, David G. Johnson. RB localizes to DNA double strand breaks and promotes DNA end resection and homologous recombination through the recruitment of SWI/SNF complex. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr PR05.

The role of CDK inhibitors in breast cancer therapy

Cyclin D1 and cyclin-dependent protein kinases CDK4/6 are part of RB-pathway which plays a very important role in the regulation of cell cycle progression and cell death. d-type cyclins associate with cdk4 and 6 to phosphorylate the Rb protein. Hyperphosphorylation of Rb promotes the release of the E2F family of transcription factors that then promotes entry into S phase through activation of key target genes. CDK inhibitors are proteins that suppress CDK-cyclin protein kinase activity in the G1 phase of the cell cycle and promote G1 arrest in response to environmental or intracellular signals. A literature search of these topics was performed through PubMed. Results from preclinical and early-stage clinical trials support the efficiency of CDK inhibitors such palbociclib, abemaciclib and ribociclib for the treatment of human cancers - including breast cancer. The first-in-class CDK4/6 inhibitor, which significantly extended PFS in combination with endocrine therapy in the first and subsequent lines of treatment for steroid receptor -positive, HER2-negative advanced breast cancer is Palbociclib. Other inhibitors (abemaciclib, ribociclib) are still in clinical trials and are a very promising group of drugs.

Abstract 4411: Cadherin engagement induces a dramatic increase in tyr-705 phosphorylation of the signal transducer and activator of transcription-3 (Stat3α) but not the dominant-negative isoform Stat3β

Abstract The purpose of this study was to examine the role of Stat3α and Stat3β in survival and apoptosis of normal and tumor cells. The Stat3 transcription factor is activated by cytokine receptors of the IL6 family, and receptor and non-receptor tyrosine kinases, plays an etiological role in neoplasia. Stat3 activation entails phosphorylation at tyr-705, Stat3 dimerization through a reciprocal, SH2 domain-phosphotyrosine interaction, and nuclear translocation. This triggers transcription of genes involved primarily in cellular survival such as survivin, MCl1 and Bcl-xL, as well as cell division such as myc. We previously demonstrated that engagement of E- or N-cadherin or cadherin-11 induces a dramatic increase in total protein levels and activity of the small GTPases, Rac and Cdc42, through inhibition of proteasomal degradation. Activated Rac leads to a surge in secretion of cytokines of the IL6 family through the transcription factor NFκB and Jak kinases, and this in turn, activates Stat3 in an autocrine manner. Stat3 inhibition in confluent, non-transformed cells induces apoptosis, pointing to a key survival role for Stat3. Full-length Stat3 (termed Stat3α) is composed of an SH2 domain, tyr-705 and a COOH terminus encoding the transcription activation domain (TAD). Stat3β is a naturally-occurring splice variant which is lacking TAD. Therefore, Stat3β dimerizes with Stat3α but is defective in transcriptional activation, resulting in inhibition of Stat3α function. Since tyr-705 is present in both isoforms, we examined the phosphorylation pattern of Stat3α vs Stat3β. Our results demonstrate that cadherin engagement brought about through confluence of non-transformed mouse fibroblasts results in phosphorylation of Stat3α-tyr705, despite the fact that the sequence around tyr-705 is the same in both isoforms. The Large Tumor antigen of Simian virus 40 oncogene (TAg) interacts with the p53 and pRb tumor-suppressors, and this leads to activation of the E2F family of transcription factors, targeting cell division genes. At the same time E2F is a potent apoptosis inducer, hence the high demand of transformed cells for antiapoptotic signals, such as Stat3. Interestingly, our data demonstrated that SVLT expression results in phosphorylation of Stat3β. Therefore, this feedback loop that reduces the activity of Stat3α, triggers apoptosis of transformed cells selectively, because of their high E2F levels. As a result, certain tumor cells which may naturally express high Stat3β levels would be very sensitive to pharmacological Stat3 inhibition, a finding which could have significant therapeutic implications. Citation Format: Zaid Taha, Stephanie Guy, Maximillian Niit, Leda Raptis, Adina Vultur, Rozanne Arulanandam. Cadherin engagement induces a dramatic increase in tyr-705 phosphorylation of the signal transducer and activator of transcription-3 (Stat3α) but not the dominant-negative isoform Stat3β. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4411.

Inhibition of Rb Phosphorylation Leads to mTORC2-Mediated Activation of Akt

The retinoblastoma (Rb) protein exerts its tumor suppressor function primarily by inhibiting the E2F family of transcription factors that govern cell-cycle progression. However, it remains largely elusive whether the hyper-phosphorylated, non-E2F1-interacting form of Rb has any physiological role. Here we report that hyper-phosphorylated Rb directly binds to and suppresses the function of mTORC2 but not mTORC1. Mechanistically, Rb, but not p107 or p130, interacts with Sin1 and blocks the access of Akt to mTORC2, leading to attenuated Akt activation and increased sensitivity to chemotherapeutic drugs. As such, inhibition of Rb phosphorylation by depleting cyclin D orusing CDK4/6 inhibitors releases Rb-mediated mTORC2 suppression. This, in turn, leads to elevated Akt activation to confer resistance to chemotherapeutic drugs in Rb-proficient cells, which can be attenuated with Akt inhibitors. Therefore, our work provides a molecular basis for the synergistic usage of CDK4/6 and Akt inhibitors in treating Rb-proficient cancer.

Ethanol extract of Kalopanax septemlobus leaf inhibits HepG2 human hepatocellular carcinoma cell proliferation via inducing cell cycle arrest at G1 phase.

To investigate the effects of an ethanol extract of Kalopanax septemlobus (Thunb.) Koidz. leaf (EEKS) on cell proliferation in human hepatocellular carcinoma cells and its mechanisms of action. Cells were treated with EEKS and subsequently analyzed for cell proliferation and flow cytometry analysis. Expressions of cell cycle regulators were determined by reverse transcriptase polymerase chain reaction analysis and Western blotting, and activation of cyclin-associated kinases studied using kinase assays. The EEKS suppressed cell proliferation in both HepG2 and Hep3B cells, but showed a more sensitive anti-proliferative activity in HepG2 cells. Flow cytometry analysis revealed an association between the growth inhibitory effect of EEKS and with G1 phase cell cycle arrest in HepG2 cells, along with the dephosphorylation of retinoblastoma protein (pRB) and enhanced binding of pRB with the E2F transcription factor family proteins. Treatment with EEKS also increased the expression of cyclin-dependent kinase (CDK) inhibitors, such as p21WAF1/CIP1 and p27KIP1, without any noticeable changes in G1 cyclins and CDKs (except for a slight decrease in CDK4). Treatment of HepG2 cells with EEKS also increased the binding of p21 and p27 with CDK4 and CDK6, which was paralleled by a marked decrease in the cyclin D- and cyclin E-associated kinase activities. Overall, our findings suggest that EEKS may be an effective treatment for liver cancer through suppression of cancer cell proliferation via G1 cell cycle arrest. Further studies are required to identify the active compounds in EEKS.

Alternate transcripts of the Drosophila “activator” E2F are necessary for maintenance of cell cycle exit during development

The E2F family of transcription factors are evolutionarily conserved regulators of the cell cycle that can be divided into two groups based on their ability to either activate or repress transcription. In Drosophila, there is only one “activator” E2F, dE2F1, which provides all of the pro-proliferative activity of E2F during development. Interestingly, the de2f1 gene can be transcribed from multiple promoters resulting in six alternate transcripts. In this study, we sought to investigate the biological significance of the alternate transcriptional start sites. We focused on the de2f1 promoter region where tissue and cell-type specific enhancer activities were observed at the larval stage. While a genomic deletion of this region, de2f1ΔRA, decreased the overall expression level of dE2F1, flies developed normally with no obvious proliferation defects. However, a detailed analysis of the de2f1ΔRA mutant eye imaginal discs revealed that dE2F1 is needed for proper cell cycle exit. We discovered that dE2F1 expression during G1 arrest prior to the differentiation process of the developing eye is important for maintaining cell cycle arrest at a later stage of the eye development. Overall, our study suggests that specific alternate transcripts of “activator” E2F, dE2F1, may have a dual function on cell cycle progression and cannot simply be viewed as a pro-proliferative transcription factor.

Structural insights into the DNA-binding specificity of E2F family transcription factors.

The mammalian cell cycle is controlled by the E2F family of transcription factors. Typical E2Fs bind to DNA as heterodimers with the related dimerization partner (DP) proteins, whereas the atypical E2Fs, E2F7 and E2F8 contain two DNA-binding domains (DBDs) and act as repressors. To understand the mechanism of repression, we have resolved the structure of E2F8 in complex with DNA at atomic resolution. We find that the first and second DBDs of E2F8 resemble the DBDs of typical E2F and DP proteins, respectively. Using molecular dynamics simulations, biochemical affinity measurements and chromatin immunoprecipitation, we further show that both atypical and typical E2Fs bind to similar DNA sequences in vitro and in vivo. Our results represent the first crystal structure of an E2F protein with two DBDs, and reveal the mechanism by which atypical E2Fs can repress canonical E2F target genes and exert their negative influence on cell cycle progression.

The Cellular Protein Complex Associated with a Transforming Region of E1A Contains c-MYC.

The cell-transforming activity of human adenovirus 5 (hAd5) E1A is mediated by the N-terminal half of E1A, which interacts with three different major cellular protein complexes, p300/CBP, TRRAP/p400, and pRb family members. Among these protein interactions, the interaction of pRb family proteins with conserved region 2 (CR2) of E1A is known to promote cell proliferation by deregulating the activities of E2F family transcription factors. The functional consequences of interaction with the other two protein complexes in regulating the transforming activity of E1A are not well defined. Here, we report that the E1A N-terminal region also interacted with the cellular proto-oncoprotein c-MYC and the homolog of enhancer of yellow 2 (ENY2). Our results suggested that these proteins interacted with an essential E1A transforming domain spanning amino acid residues 26 to 35 which also interacted with TRRAP and p400. Small interfering RNA (siRNA)-mediated depletion of TRRAP reduced c-MYC interaction with E1A, while p400 depletion did not. In contrast, depletion of TRRAP enhanced ENY2 interaction with E1A, suggesting that ENY2 and TRRAP may interact with E1A in a competitive manner. The same E1A region additionally interacted with the constituents of a deubiquitinase complex consisting of USP22, ATXN7, and ATXN7L3 via TRRAP. Acute short hairpin RNA (shRNA)-mediated depletion of c-MYC reduced the E1A transforming activity, while depletion of ENY2 and MAX did not. These results suggested that the association of c-MYC with E1A may, at least partially, play a role in the E1A transformation activity, independently of MAX. The transforming region of adenovirus E1A consists of three short modules which complex with different cellular protein complexes. The mechanism by which one of the transforming modules, CR2, promotes cell proliferation, through inactivating the activities of the pRb family proteins, is better understood than the activities of the other domains. Our analysis of the E1A proteome revealed the presence of the proto-oncoprotein c-MYC and of ENY2. We mapped these interactions to a critical transforming module of E1A that was previously known to interact with the scaffolding molecule TRRAP and the E1A-binding protein p400. We showed that c-MYC interacted with E1A through TRRAP, while ENY2 interacted with it independently. The data reported here indicated that depletion of c-MYC in normal human cells reduced the transforming activity of E1A. Our result raises a novel paradigm in oncogenic transformation by a DNA viral oncogene, the E1A gene, that may exploit the activity of a cellular oncogene, the c-MYC gene, in addition to inactivation of the tumor suppressors, such as pRb.

Conserved E2F mediated metastasis in mouse models of breast cancer and HER2 positive patients.

To improve breast cancer patient outcome work must be done to understand and block tumor metastasis. This study leverages bioinformatics techniques and traditional genetic screens to create a novel method of discovering potential contributors of tumor progression with a focus on tumor metastasis. A database of 1172 of expression data from a variety of mouse models of breast cancer was assembled and queried using previously defined oncogenic activity signatures. This analysis revealed high activity of the E2F family of transcription factors in the MMTV-Neu mouse model. A genetic cross of MMTV-Neu mice into an E2F1 null, E2F2 null, or E2F3 heterozygous background revealed significant changes in tumor progression specifically reductions in tumor latency and metastasis with E2F1 or E2F2 loss. These findings were found to be conserved in human HER2 positive patients. Patients with high E2F1 activity were shown to have worse outcomes such as relapse free survival and distant metastasis free survival. This study shows conserved mechanisms of tumor progression in human breast cancer subtypes and analogous mouse models and underlies the importance of increased research into the characterization of and comparisons between mouse and human tumors to identify which mouse models resemble each subtype of human breast cancer.