E2F-responsive Genes Research Articles

Abstract 1726: Structure-function analysis of retinoblastoma tumor suppressor protein in human cone precursors

Abstract Background: The retinoblastoma protein, pRB, is a key cell cycle governor that is functionally impaired in most cancers, but its mechanism of suppressing tumorigenesis in a tumor cell-of-origin has not been characterized. This study aims to define pRB structural features and corresponding cellular functions that suppress cone precursor (CP) proliferation, likely the first step in retinoblastomagenesis. Method: Intact human fetal retinae were co-transduced with lentiviruses to knockdown endogenous pRB and ectopically express wild type or structure-based substitution mutant pRBs. The ectopically expressed pRB mutants had selectively impaired interactions with E2F transactivation domains (ΔG), with the E2F1 “marked box” domain (ΔS), or with the LxCxE protein interaction motif (ΔL). At day 12-75 post-infection, transduced retinae were analyzed by quantitative immunofluorescence staining of Ki67/EdU, pRB and CP marker L/M-opsin. Results: In intact retina, pRB knockdown induced cell cycle re-entry of L/M-opsin+ postmitotic cone precursors, whereas those reconstituted with wild type pRB did not express Ki67 indicative of a block to cell cycle entry. Unexpectedly, all three mutants (pRB-ΔG, -ΔL, and -ΔS) were able to block cell cycle re-entry when expressed at the high-normal range the of endogenous pRB. However, at the low-normal expression level, ectopic wild type but not mutant pRBs suppressed proliferation. Conclusion: All three surface targeted mutants (pRB-ΔG, ΔL, and ΔS) retain partial ability to suppress cone precursor cell cycle entry. This may be attributed to the presence of multiple independent cell cycle inhibitory functions. These findings suggest that pRB-mediated regulation of E2F-responsive cell cycle genes, interactions with LxCxE motif-containing proteins, and EZH2 mediated repression of repetitive elements are individually dispensable for suppressing aberrant cone precursor proliferation. Citation Format: Sijia Wang, Brendan Grubbs, Matthew Thornton, David Cobrinik. Structure-function analysis of retinoblastoma tumor suppressor protein in human cone precursors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1726.

Human cytomegalovirus-encoded viral cyclin-dependent kinase (v-CDK) UL97 phosphorylates and inactivates the retinoblastoma protein-related p107 and p130 proteins

The human cytomegalovirus (HCMV)-encoded viral cyclin-dependent kinase (v-CDK) UL97 phosphorylates the retinoblastoma (Rb) tumor suppressor. Here, we identify the other Rb family members p107 and p130 as novel targets of UL97. UL97 phosphorylates p107 and p130 thereby inhibiting their ability to repress the E2F-responsive E2F1 promoter. As with Rb, this phosphorylation, and the rescue of E2F-responsive transcription, is dependent on the L1 LXCXE motif in UL97 and its interacting clefts on p107 and p130. Interestingly, UL97 does not induce the disruption of all p107-E2F or p130-E2F complexes, as it does to Rb-E2F complexes. UL97 strongly interacts with p107 but not Rb or p130. Thus the inhibitory mechanisms of UL97 for Rb family protein-mediated repression of E2F-responsive transcription appear to differ for each of the Rb family proteins. The immediate early 1 (IE1) protein of HCMV also rescues p107- and p130-mediated repression of E2F-responsive gene expression, but it does not induce their phosphorylation and does not disrupt p107-E2F or p130-E2F complexes. The unique regulation of Rb family proteins by HCMV UL97 and IE1 attests to the importance of modulating Rb family protein function in HCMV-infected cells.

Abstract IA21: Mitogenic signaling and the RB/p53 network

Abstract Cells entering the division cycle from a quiescent state (G0) in response to mitogenic cues synthesize one or more D-type cyclins (D1, D2, D3) that assemble in G1 phase with cyclin-dependent kinases CDK4 and/or CDK6. Notably, the transcription, assembly with CDK 4/6, and stability of D-type cyclins are each mitogen-dependent steps. Accumulation of cyclin D-dependent kinases in G1 phase leads to the progressive phosphorylation of the retinoblastoma protein (RB), facilitating transcription of E2F-responsive genes, including cyclins E and A, whose CDK2-dependent functions are normally required in S phase. Moreover, the stoichiometric sequestration of CDK2 inhibitors, p27Kip1 and p21Cip1, by cyclin D-dependent kinases, further enables CDK2 activation as well as SCFSkp2-dependent p27 degradation as cells approach the G1/S boundary. In G0, RB remains unphosphorylated, and E2F-responsive genes are repressed by the DREAM complex, which includes E2F-4/5, at least one retinoblastoma family member (RBL2/p130), and the MuvB complex. An open question is whether CDK4 and CDK6 are responsible, at least in part, for dissolution of the DREAM complex or relief of p130/E2F repression in early G1 phase. Cells in G0 and G1 express the APCCDH1 E3 ubiquitin ligase that prevents accumulation of the mitotic cyclins, A and B, prior to S phase entry. As cells near the G1/S transition, APCCDH1 is inactivated by CDK2 in conjunction with the E2F-responsive early mitotic inhibitor EMI1. Cyclin D-CDK4/6 complexes accumulate maximally in the nucleus at G1/S, where their phosphorylation by GSK-3β triggers their subsequent nuclear export, ubiquitination, and proteasomal degradation in S phase. In continuously cycling cells, Ras signaling in G2 phase restores nuclear cyclin D, enabling its re-accumulation before M phase; cyclin D-CDK4/6 activity in G2/M shortens the subsequent G1 interval and cell cycle length. Mitogen withdrawal, leads to rapid cyclin D turnover and ultimately to G1/G0 arrest. Similar effects can result from oncogene-mediated induction of the specific CDK4/6 inhibitor p16INK4A, or by CDK4/6 inhibitory drugs (CDK4/6i's) that act as chemical p16 mimetics. Notably, continuous CDK4/6 inhibition by CDK4/6i's or p16 can induce reversible (quiescence) or irreversible (senescence) cell cycle exit; understanding the factors that distinguish these outcomes continues to represent a challenge. Although monotherapy with orally available, potent, and specific CDK4/6i's has not generally yielded durable clinical benefits in cancer treatment, these drugs can induce significant progression-free survival with minimal dose limiting side effects (principally clinically manageable neutropenia) when combined with targeted inhibitors of mitogenic signaling pathways that limit the accumulation of D-type cyclins. Perhaps, an “addiction” of cancer cells to mitogenic pathways constitutively activated by oncogenic mutations increases their vulnerability to CDK4/6i's while sparing normal cycling cells. Expression of the CDKN2A/B gene cluster (encoding p16, p15INK4B, and ARF) is driven by an upstream super-enhancer. Canonical super-enhancer histone “marks” (H3K4me1 and H3K27Ac) are detected in cells that are able to express CDKN2A/B in response to hyperproliferative signals but are absent from their reprogrammed iPS derivatives or from ES cells. More than 30 independent GWAS studies have fingered SNPs within the CDKN2A/B super-enhancer region that are associated not only with various cancers but also with common degenerative diseases of aging populations, including coronary artery disease, aortic aneurysm, and type II diabetes. These findings suggest that while downregulation of CDKN2A/B results in a loss of tumor suppression, its increased expression during aging leads to cellular senescence and a loss of tissue regenerative capacity. Citation Format: Charles J. Sherr. Mitogenic signaling and the RB/p53 network. [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 IA21.

MiR-137 Modulates a Tumor Suppressor Network-Inducing Senescence in Pancreatic Cancer Cells.

Activating K-Ras mutations occurs frequently in pancreatic cancers and is implicated in their development. Cancer-initiating events, such as oncogenic Ras activation, lead to the induction of cellular senescence, a tumor suppressor response. During senescence, the decreased levels of KDM4A lysine demethylase contribute to p53 activation, however, the mechanism by which KDM4A is downregulated is unknown. We show that miR-137 targets KDM4A mRNA during Ras-induced senescence and activates both p53 and retinoblastoma (pRb) tumor suppressor pathways. Restoring the KDM4A expression contributed to bypass of miR-137-induced senescence and inhibition of endogenous miR-137 with an miRNA sponge-compromised Ras-induced senescence. miR-137 levels are significantly reduced in human pancreatic tumors, consistent with previous studies revealing a defective senescence response in this cancer type. Restoration of miR-137 expression inhibited proliferation and promoted senescence of pancreatic cancer cells. These results suggest that modulating levels of miR-137 may be important for triggering tumor suppressor networks in pancreatic cancer.

Abstract P2-04-03: SUMO wrestles breast cancer: SUMO posttranslational modification directs breast cancer cell epigenome

Abstract Epigenetic reprogramming allows breast cancer (BCa) cells to bypass normal growth checkpoints and acquire an aggressive morphology. Function of critical epigenetic proteins is directed by small ubiquitin-like modifiers (SUMO) posttranslational modification (PTM). Components of the SUMO system are dysregulated in BCa. Our objective is to delineate how changes to SUMO-PTM affect epigenetic programs that coordinate BCa pathology. We report that chromatin-bound SUMO protease SENP7L is upregulated in metastatic tamoxifen-resistant (TamR) and triple-negative BCa. SENP7L facilitates chromatin remodeling that potentiates the growth and invasiveness of these BCa subtypes. SENP7L interacts with and deSUMOylates heterochromatin protein 1 alpha (HP1-alpha/CBX5) to mediates these responses. Canonical models suggest that HP1-alpha reads the histone mark to localize predominantly at constitutive heterochromatin. Our studies challenge the old paradigm as we show that SUMOylated HP1-alpha is enriched outside heterochromatin at euchromatin, including E2F-responsive and mesenchymal gene loci. Recruitment of SUMOylated HP1-alpha supports de novo heterochromatin formation at these loci. However, SUMOylated HP1-alpha is lost in aggressive BCa due to SENP7L induction. Consistently, SENP7L upregulation prompts chromatin de-condensation and gene transcription. Nuclei acid and chemical targeting of SENP7L reduces self-renewal/differentiation properties of TamR mammospheres as well as tumor volume and secondary tumor formation of TamR cells. Hence, SENP7L inhibitors can serve as promising tools to modulate epigenetic abnormalities in TamR BCa. Citation Format: Bawa-Khalfe T, Kumar S, Lin F-M. SUMO wrestles breast cancer: SUMO posttranslational modification directs breast cancer cell epigenome. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-04-03.

CDK4 Amplification Reduces Sensitivity to CDK4/6 Inhibition in Fusion-Positive Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and includes a PAX3- or PAX7-FOXO1 fusion-positive subtype. Amplification of chromosomal region 12q13-q14, which contains the CDK4 proto-oncogene, was identified in an aggressive subset of fusion-positive RMS. CDK4/6 inhibitors have antiproliferative activity in CDK4-amplified liposarcoma and neuroblastoma, suggesting CDK4/6 inhibition as a potential therapeutic strategy in fusion-positive RMS. We examined the biologic consequences of CDK4 knockdown, CDK4 overexpression, and pharmacologic CDK4/6 inhibition by LEE011 in fusion-positive RMS cell lines and xenografts. Knockdown of CDK4 abrogated proliferation and transformation of 12q13-14-amplified and nonamplified fusion-positive RMS cells via G1-phase cell-cycle arrest. This arrest was mediated by reduced RB phosphorylation and E2F-responsive gene expression. Significant differences in E2F target expression, cell-cycle distribution, proliferation, or transformation were not observed in RMS cells overexpressing CDK4. Treatment with LEE011 phenocopied CDK4 knockdown, decreasing viability, RB phosphorylation, and E2F-responsive gene expression and inducing G1-phase cell-cycle arrest. Although all fusion-positive cell lines showed sensitivity to CDK4/6 inhibition, there was diminished sensitivity associated with CDK4 amplification and overexpression. This variable responsiveness to LEE011 was recapitulated in xenograft models of CDK4-amplified and nonamplified fusion-positive RMS. Our data demonstrate that CDK4 is necessary but overexpression is not sufficient for RB-E2F-mediated G1-phase cell-cycle progression, proliferation, and transformation in fusion-positive RMS. Our studies indicate that LEE011 is active in the setting of fusion-positive RMS and suggest that low CDK4-expressing fusion-positive tumors may be particularly susceptible to CDK4/6 inhibition.

Abstract 3093: CDK4 amplification reduces sensitivity to CDK4/6 inhibition in fusion-positive rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and includes an aggressive, PAX3-FOXO1 fusion-positive subtype. Amplification of chromosomal region 12q13-q14, which contains the CDK4 proto-oncogene, was identified in a subset of fusion-positive RMS. Other tumor types with CDK4 amplification or overexpression, including liposarcoma and neuroblastoma, are sensitive to CDK4/6 inhibition, suggesting that CDK4/6-targeted therapies may provide a new treatment strategy in fusion-positive RMS. To evaluate the role of CDK4 in chromosomal region 12q13-14 amplification in fusion-positive RMS and the potential clinical utility of CDK4/6 inhibition in this disease setting, we examined the biological consequences of CDK4 knockdown, CDK4 overexpression, and pharmacologic CDK4/6 inhibition in fusion-positive RMS in vitro and in vivo. Knockdown of CDK4 abrogated proliferation and transformation of 12q13-14-amplified and non-amplified fusion-positive RMS cells via G1-phase cell cycle arrest. This arrest was associated with reduced RB phosphorylation and E2F-responsive gene expression. Significant differences in E2F target expression, however, were not observed in RMS cells overexpressing CDK4 or in fusion-positive tumors harboring 12q13-14 amplification relative to control cells or tumors lacking amplification, respectively. Treatment with the small molecule CDK4/6 inhibitor LEE011 phenocopied CDK4 knockdown, decreasing viability, RB phosphorylation, and E2F-responsive gene expression and inducing G1-phase cell cycle arrest. All fusion-positive RMS cell lines showed sensitivity to CDK4/6 inhibition, with evidence of differential antitumor activity resulting from an inverse relationship between CDK4 expression and inhibitor vulnerability. This variable responsiveness to LEE011 was recapitulated in xenograft models of CDK4-amplified and non-amplified fusion-positive RMS. Our findings demonstrate that CDK4 is necessary for RB-E2F-mediated G1-phase cell cycle progression, proliferation, and transformation in fusion-positive RMS regardless of CDK4 amplification status. Our studies further indicate that single-agent LEE011 is active in the setting of fusion-positive RMS and suggest that CDK4 amplification may be a marker of reduced sensitivity whereas low CDK4 expression may be associated with higher susceptibility to CDK4/6 inhibition. Collectively, our data provide preclinical evidence supporting further investigation of CDK4/6-targeted therapies in treatment regimens for fusion-positive RMS. Citation Format: Mary E. Olanich, Wenyue Sun, Stephen M. Hewitt, Zied Abdullaev, Svetlana D. Pack, Frederic G. Barr. CDK4 amplification reduces sensitivity to CDK4/6 inhibition in fusion-positive rhabdomyosarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3093. doi:10.1158/1538-7445.AM2015-3093

Molecular Determinants for the Inactivation of the Retinoblastoma Tumor Suppressor by the Viral Cyclin-dependent Kinase UL97

The retinoblastoma (Rb) tumor suppressor restricts cell cycle progression by repressing E2F-responsive transcription. Cellular cyclin-dependent kinase (CDK)-mediated Rb inactivation through phosphorylation disrupts Rb-E2F complexes, stimulating transcription. The human cytomegalovirus (HCMV) UL97 protein is a viral CDK (v-CDK) that phosphorylates Rb. Here we show that UL97 phosphorylates 11 of the 16 consensus CDK sites in Rb. A cleft within Rb accommodates peptides with the amino acid sequence LXCXE. UL97 contains three such motifs. We determined that the first LXCXE motif (L1) of UL97 and the Rb cleft enhance UL97-mediated Rb phosphorylation. A UL97 mutant with a non-functional L1 motif (UL97-L1m) displayed significantly reduced Rb phosphorylation at multiple sites. Curiously, however, it efficiently disrupted Rb-E2F complexes but failed to relieve Rb-mediated repression of E2F reporter constructs. The HCMV immediate early 1 protein cooperated with UL97-L1m to inactivate Rb in transfection assays, likely indicating that cells infected with a UL97-L1m mutant virus show no defects in growth or E2F-responsive gene expression because of redundant viral mechanisms to inactivate Rb. Our data suggest that UL97 possesses a mechanism to elicit E2F-dependent gene expression distinct from disruption of Rb-E2F complexes and dependent upon both the L1 motif of UL97 and the cleft region of Rb.

Tumorigenic activity of merkel cell polyomavirus T antigens expressed in the stratified epithelium of mice.

Merkel cell polyomavirus (MCPyV) is frequently associated with Merkel cell carcinoma (MCC), a highly aggressive neuroendocrine skin cancer. Most MCC tumors contain integrated copies of the viral genome with persistent expression of the MCPyV large T (LT) and small T (ST) antigen. MCPyV isolated from MCC typically contains wild-type ST but truncated forms of LT that retain the N-terminus but delete the C-terminus and render LT incapable of supporting virus replication. To determine the oncogenic activity of MCC tumor-derived T antigens in vivo, a conditional, tissue-specific mouse model was developed. Keratin 14-mediated Cre recombinase expression induced expression of MCPyV T antigens in stratified squamous epithelial cells and Merkel cells of the skin epidermis. Mice expressing MCPyV T antigens developed hyperplasia, hyperkeratosis, and acanthosis of the skin with additional abnormalities in whisker pads, footpads, and eyes. Nearly half of the mice also developed cutaneous papillomas. Evidence for neoplastic progression within stratified epithelia included increased cellular proliferation, unscheduled DNA synthesis, increased E2F-responsive genes levels, disrupted differentiation, and presence of a DNA damage response. These results indicate that MCPyV T antigens are tumorigenic in vivo, consistent with their suspected etiologic role in human cancer.

Abstract LB-39: Human cone precursor-derived retinoblastoma tumors

Abstract Retinoblastomas initiate in response to biallelic RB1 inactivation and loss of functional Rb protein, yet the cell of origin and cellular circuitry that sensitizes to Rb loss have been unclear. Here, we probed the cellular origin of retinoblastoma by assessing cell type-specific responses to shRNA-mediated Rb depletion in dissociated fetal retina, in intact retina, and in isolated cone precursor populations. In dissociated retinal cultures, RB1 knockdown induced S phase entry and proliferation of cells expressing markers of cone precursors (CRX + L/M opsin, or CRX + cone arrestin, or CRX + RXRγ) but not markers or other retinal cell types. Similarly, RB1 knockdown induced cone precursor proliferation in the undissociated retina and in prospectively isolated cone precursor populations. Proliferation of FACS-isolated cone precursors correlated with the induction of E2F-responsive genes and depended upon factors having strong cone precursor expression and crucial roles in retinoblastoma cell proliferation, including MDM2, MYCN, TRβ2, and SKP2. The proliferative response to Rb depletion was enhanced by co-depletion of the Rb-related p130 but was abrogated by co-depletion of p107, concordant with frequent p130 but not p107 losses in retinoblastoma tumors. Rb- and Rb/p130-depleted cone precursors proliferated in suspension and formed tumors in orthotopic xenografts with histologic features and protein expression profiles typical of differentiated human retinoblastomas. These findings indicate that post-mitotic human cone precursors are uniquely sensitive to the proliferative and oncogenic effects of Rb depletion and imply that human cone precursor circuitry collaborates with inactivating RB1 mutations to initiate retinoblastoma tumorigenesis. Citation Format: Xiaoliang L. Xu, Hardeep P. Singh, Lu Wang, Donglai Qi, Bradford K. Poulos, David H. Abramson, Suresh C. Jhanwar, David Cobrinik. Human cone precursor-derived retinoblastoma tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-39. doi:10.1158/1538-7445.AM2014-LB-39

A Novel Subset of Human Tumors That Simultaneously Overexpress Multiple E2F-responsive Genes Found in Breast, Ovarian, and Prostate Cancers.

Reasoning that overexpression of multiple E2F-responsive genes might be a useful marker for RB1 dysfunction, we compiled a list of E2F-responsive genes from the literature and evaluated their expression in publicly available gene expression microarray data of patients with breast cancer, serous ovarian cancer, and prostate cancer. In breast cancer, a group of tumors was identified, each of which simultaneously overexpressed multiple E2F-responsive genes. Seventy percent of these genes were concerned with cell cycle progression, DNA repair, or mitosis. These E2F-responsive gene overexpressing (ERGO) tumors frequently exhibited additional evidence of Rb/E2F axis dysfunction, were mostly triple negative, and preferentially overexpressed multiple basal cytokeratins, suggesting that they overlapped substantially with the basal-like tumor subset. ERGO tumors were also identified in serous ovarian cancer and prostate cancer. In these cancer types, there was no evidence for a tumor subset comparable to the breast cancer basal-like subset. A core group of about 30 E2F-responsive genes were overexpressed in all three cancer types. Thus, it appears that disorders of the Rb/E2F axis can arise at multiple organ sites and produce tumors that simultaneously overexpress multiple E2F-responsive genes.

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.

Tumor suppressor role of chromatin-remodeling factor ARID1A

The mammalian SWI/SNF complex is one of ATP-dependent chromatin-remodeling complexes, which plays important roles in cell proliferation, differentiation, development and tumor suppression. ARID1A (AT-rich interactive domain-containing protein 1A) is a large subunit of SWI/SNF complex, and also an ARID family member with non- sequence-specific DNA binding activity. ARID1A is a tumor suppressor gene which is frequently mutated in many cancers, such as ovarian, bladder and gastric cancers. ARID1A can suppress cell proliferation through the up-regulation of p21 and the down-regulation of E2F-responsive genes. These findings on ARID1A and its role of tumor suppression contribute to understanding the mechanism of cancer development and developing new therapy for cancer.It is introduced in the review that ARID1A basic characteristic, related to cancer development, and biological role for full understanding of ARID1A.

The Nuclear Protein ALY Binds to and Modulates the Activity of Transcription Factor E2F2

E2F transcription factors control the expression of genes involved in a variety of essential cellular processes and consequently their activity needs to be tightly regulated. Protein-protein interactions are thought to be key modulators of E2F activity. To gain insight into the mechanisms that regulate the activity of E2F2, we searched for novel proteins that associate with this transcription factor. We show that the nuclear protein ALY (THO complex 4), originally described as a transcriptional co-activator, associates with DNA-bound E2F2 and represses its transcriptional activity. The capacity of ALY to modulate gene expression was analyzed with expression microarrays by characterizing the transcriptome of E2F2 expressing HEK293T cells in which ALY was either overexpressed or silenced. We show that ALY influences the expression of more than 400 genes, including 98 genes bearing consensus E2F motifs. Thus, ALY emerges as a novel E2F2-interacting protein and a relevant modulator of E2F-responsive gene expression.

E2F6 Associates with BRG1 in Transcriptional Regulation

The E2F6 protein functions as an Rb-independent repressor of gene transcription. We have previously provided evidence suggesting a role for E2F6 in repression of E2F-responsive genes at S phase. Here, we have identified BRG1, the ATPase subunit of the SWI/SNF chromatin-remodeling complex, as an E2F6 interacting protein. Immunoprecipitation experiments demonstrate that BRG1 binds specifically to E2F6 and E2F4 but not the activator E2Fs. E2F6 was also able to interact with BAF155, a BRG1-associated factor, in the SWI/SNF complex. Chromatin immunoprecipitation assays demonstrate the binding of BRG1 coincident with E2F6 on G1/S gene promoters during S phase. Collectively, our studies suggest that E2F6 may recruit BRG1 in transcriptional regulation of genes important for G1/S phase transition of the cell cycle.

Differential expression of SUMO-specific protease 7 variants regulates epithelial–mesenchymal transition

Two Sentrin/small ubiquitin-like modifier (SUMO)-specific protease 7 (SENP7) variants are naturally expressed in breast epithelia. Breast cancer (BCa) onset down-regulates the short SENP7 splice variant (SENP7S) and enhances the long transcript (SENP7L). Here, we show that SENP7L induction promotes gene expression profiles that favor aberrant proliferation and initiate epithelial-mesenchymal transition (EMT). SENP7L exhibits an interaction domain for the epigenetic remodeler heterochromatin protein 1 α (HP1α) and isopeptidase activity against SUMO-modified HP1α. Loss of this interaction domain, as observed with SENP7S, favors HP1α SUMOylation. SUMOylated HP1α is enriched at E2F-responsive and mesenchymal gene promoters, silences transcription of these genes, and promotes cellular senescence. Elevated SENP7L renders HP1α hypo-SUMOylated, which relieves transcriptional repression of the same genes and concurrently decreases transcription of epithelial-promoting genes via an HP1α-independent mechanism. Consequently, SENP7L levels correlate with EMT, motility, and invasiveness of BCa cells. Stable knockdown of elevated SENP7L levels lessens the dissemination of highly metastatic BCa cells to the lungs from primary implantation sites in in vivo studies. Thus, differential splicing of the SENP7 regulates either tumor suppression or progression.

Mechanism of action of ingenol mebutate gel in reconstructed human skin assessed by transcriptomics.

e19056 Background: Ingenol mebutate (IM) gel topical applied once daily for 2-3 consecutive days, has recently been approved by FDA in treating actinic keratoses (AK), as a safe, tolerable and effective treatment. IM, a highly active biological diterpene ester, has shown in vitro and in vivo to induce a dual mechanism of action characterized by, first, cell death induced at high micromolar concentrations and, second, a protein kinase C dependent immune activation in the nanomolar range. The aim of this study was to investigate the mechanism of action of IM in reconstructed human skin (RHS). Methods: IM 0.05% gel or vehicle was applied topically to RHS (EpiDermFT) and normal pigskin and IM concentrations were measured after 2 and 24 hrs. In RHS, biopsies were obtained 1, 6 and 24 hrs after treatment and global transcriptomic profiling (Affymetrix HG 1.0 ST) was assessed. The analysis included transcription factor binding site (TFBS) and gene set enrichment analysis (GSEA). Results: We compared IM’s penetration through normal skin to RHS and found a 10-fold higher permeability in RHS, which likely models the increased permeability of AK lesions. Consequently we further investigated IM’s effects on RHS. Based on GSEA, we identified the major effects of IM to comprise: cell death, inflammation and wound healing responses, indicated by differential expression of e.g. caspases, DAPK1, chemokine ligands, HAS2, MMPs, SERPINs, ILs (and their receptors). A TFBS analysis identified E2F regulated genes as overrepresented, albeit in opposite directions in dermis (up-regulated) compared to epidermis (down-regulated). IM transiently activates PKC, and via this activation causes transcriptional silencing of E2F-responsive genes. The differential effect on dermis and epidermis could be due to different levels of PKC isoforms in the different skin layers. Indeed, a specific, inflammatory response was observed and several genes, including MMP10 and IL1R2, displayed a time-dependent increase in expression, reaching a maximum at 24h. Conclusions: In conclusion, IM displays pleiotropic effects, of which many are identified on a transcriptional level. Our data suggest involvement of in particular PKC, ERK2, and E2F regulated genes.

The E2F6 repressor activates gene expression in myocardium resulting in dilated cardiomyopathy

The E2F/Rb pathway regulates cardiac growth and development and holds great potential as a therapeutic target. The E2F6 repressor is a unique E2F member that acts independently of pocket proteins. Forced expression of E2F6 in mouse myocardium induced heart failure and mortality, with severity of symptoms correlating to E2F6 levels. Echocardiography demonstrated a 37% increase (P<0.05) in left ventricular end-diastolic diameter and reduced ejection fraction (<40%, P<0.05) in young transgenic (Tg) mice. Microarray and qPCR analysis revealed a paradoxical increase in E2F-responsive genes, which regulate the cell cycle, without changes in cardiomyocyte cell number or size in Tg mice. Young adult Tg mice displayed a 75% (P<0.01) decrease in gap junction protein connexin-43, resulting in abnormal electrocardiogram including a 24% (P<0.05) increase in PR interval. Further, mir-206, which targets connexin-43, was up-regulated 10-fold (P<0.05) in Tg myocardium. The mitogen-activated protein kinase pathway, which regulates the levels of miR-206 and connexin-43, was activated in Tg hearts. Thus, deregulated E2F6 levels evoked abnormal gene expression at transcriptional and post-transcriptional levels, leading to cardiac remodeling and dilated cardiomyopathy. The data highlight an unprecedented role for the strict regulation of the E2F pathway in normal postnatal cardiac function.

Adenovirus E1A Directly Targets the E2F/DP-1 Complex

Deregulation of the cell cycle is of paramount importance during adenovirus infection. Adenovirus normally infects quiescent cells and must initiate the cell cycle in order to propagate itself. The pRb family of proteins controls entry into the cell cycle by interacting with and repressing transcriptional activation by the E2F transcription factors. The viral E1A proteins indirectly activate E2F-dependent transcription and cell cycle entry, in part, by interacting with pRb and family members to free the E2Fs. We report here that an E1A 13S isoform can unexpectedly activate E2F-responsive gene expression independently of binding to the pRb family of proteins. We demonstrate that E1A binds to E2F/DP-1 complexes through a direct interaction with DP-1. E1A appears to utilize this binding to recruit itself to E2F-regulated promoters, and this allows the E1A 13S protein, but not the E1A 12S protein, to activate transcription independently of interaction with pRb. Importantly, expression of E1A 13S, but not E1A 12S, led to significant enhancement of E2F4 occupancy of E2F sites of two E2F-regulated promoters. These observations identify a novel mechanism by which adenovirus deregulates the cell cycle and suggest that E1A 13S may selectively activate a subset of E2F-regulated cellular genes during infection.

Abstract 5048: Targeting regulators of Chk1 to enhance cytotoxic efficacy of HDAC inhibitors

Abstract Introduction: Predicting and overcoming drug resistance is a major difficulty facing the efficacy of drugs in clinical trials. Histone deacetylase inhibitors (HDACis) are known to induce cell death and have been used in clinic trials as anticancer drugs, however, the cytotoxic effects of HDACis remains to be limited with no clear mechanism of resistance has been elucidated to explain this limited efficacy. In this study we showed that downregulation of the G2 checkpoint kinase 1 (Chk1) plays an essential role in inducing cell death by HDACis. We identified E2F as a main regulator of Chk1 expression in cancer cells, suggesting that inhibition of Chk1 through E2F may enhance the cytotoxic effects of HDACis in clinical studies. Methods: A549, H1299, PC9, HCT116, HT1080, MNNG, H2172, Saos-2, MG63, U2OS, p21-/- and p21+/+ MEF cell lines were grown in appropriate medium and treated with the HDAC inhibitors, SaHa, LBH589 or MS275. Western blot and qRT-PCR experiments were performed to assess the effect of HDACis on Chk1/G2-M checkpoint pathways and on E2F-mediated gene expression profiles. Additionally, knock out and gain of function experiments were done to better understand the interaction between Chk1, E2F and p21Waf1 in HDACi mediated cell death. The results of the in vitro cell lines studies were corroborated in the ex vivo experiments with patient tumor tissue. Results: We showed that HDACi treatment leads to inhibition of Chk1 expression and consequently to increased activity of CDC25 and CDC2 (CDK1) enzymes causing premature mitotic entry and cell death. In the knock-down experiments we found that inhibition of E2F1 or E2F3 leads to reduction of Chk1 expression and potentiation of cell death by HDACis. Similar results were observed using patient-derived tumor samples treated with SaHa and an E2F inhibitor (HLM006474). Additionally, over-expression of E2F1 or E2F3 dramatically decreased HDACi-mediated cell death. In time course experiments, Chk1 as well as other E2F-responsive genes were down regulated after HDACi treatment accompanied by an expected p21Waf1 induction. In p21Waf1 +/+ and -/- MEFs we showed that lack of p21Waf1 results in increased apoptosis, suggesting that early induction of p21Waf1 by HDACis negatively regulates HDACi-induced cell death, which likely contributes to HDACi resistance in tumor cells by preventing cells to undergo mitotic cell death. Conclusions: Our results demonstrate that E2F-mediated downregulation of Chk1 plays a key role in HDACi-induced cell. We provide evidence that lack of Chk1 downregulation and activation of CDC2 is associated with resistance to HDACis indicating that Chk1 may represent a clinically relevant biomarker to assess the efficacy of HDACis in patient tumor samples. Taken together our data lends insight into the molecular mechanism of sensitivity and resistance to HDACi-induced cell death and will hopefully provide a stronger foundation for future clinical translation with HDACis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5048. doi:10.1158/1538-7445.AM2011-5048