Loss of E2F1 Extends Survival and Accelerates Oral Tumor Growth in HPV-Positive Mice

The Human Papillomavirus (HPV) is associated with several human cancers, including head and neck squamous cell carcinomas (HNSCCs). HPV expresses the viral oncogene E7 that binds to the retinoblastoma protein (RB1) in order to activate the E2F pathway. RB1 can mediate contradictory pathways—cell growth and cell death via E2F family members. Here, we assessed the extent to which E2F1 mediates lethality of HPV oncogenes. Ubiquitous expression of the HPV oncogenes E6 and E7 caused lethality in mice that was associated with focal necrosis in hepatocytes and pancreatic tissues. Furthermore, all organs expressing HPV oncogenes displayed up-regulation of several E2F1 target genes. The E2F1 pathway mediated lethality in HPV-positive mice because deletion of E2F1 increased survival of mice ubiquitously expressing HPV oncogenes. E2F1 similarly functioned as a tumor suppressor in HPV-positive oral tumors as tumors grew faster with homozygous loss of E2F1 compared to tumors with heterozygous loss of E2F1. Re-expression of E2F1 caused decreased clonogenicity in HPV-positive cancer cells. Our results indicate that HPV oncogenes activated the E2F1 pathway to cause lethality in normal mice and to suppress oral tumor growth. These results suggest that selective modulation of the E2F1 pathway, which is activated in HPV tumors, may facilitate tumor regression

A High-Throughput Cell-Based Screen Identified a 2-[(E)-2-Phenylvinyl]-8-Quinolinol Core Structure That Activates p53

p53 function is frequently inhibited in cancer either through mutations or by increased degradation via MDM2 and/or E6AP E3-ubiquitin ligases. Most agents that restore p53 expression act by binding MDM2 or E6AP to prevent p53 degradation. However, fewer compounds directly bind to and activate p53. Here, we identified compounds that shared a core structure that bound p53, caused nuclear localization of p53 and caused cell death. To identify these compounds, we developed a novel cell-based screen to redirect p53 degradation to the Skip-Cullin-F-box (SCF) ubiquitin ligase complex in cells expressing high levels of p53. In a multiplexed assay, we coupled p53 targeted degradation with Rb1 targeted degradation in order to identify compounds that prevented p53 degradation while not inhibiting degradation through the SCF complex or other proteolytic machinery. High-throughput screening identified several leads that shared a common 2-[(E)-2-phenylvinyl]-8-quinolinol core structure that stabilized p53. Surface plasmon resonance analysis indicated that these compounds bound p53 with a KD of 200 ± 52 nM. Furthermore, these compounds increased p53 nuclear localization and transcription of the p53 target genes PUMA, BAX, p21 and FAS in cancer cells. Although p53-null cells had a 2.5±0.5-fold greater viability compared to p53 wild type cells after treatment with core compounds, loss of p53 did not completely rescue cell viability suggesting that compounds may target both p53-dependent and p53-independent pathways to inhibit cell proliferation. Thus, we present a novel, cell-based high-throughput screen to identify a 2-[(E)-2-phenylvinyl]-8-quinolinol core structure that bound to p53 and increased p53 activity in cancer cells. These compounds may serve as anti-neoplastic agents in part by targeting p53 as well as other potential pathways

The common 2-[(E)-2-phenylvinyl]-8-quinolinol core structure bound p53 and disrupted p53 degradation.

<p>(A) Of the 269 potential compounds that increased p53-Luc activity by ≥2-fold above background, 6 structures shared common core 2-[(E)-2-phenylvinyl]-8-quinolinol structure. (B) Structure activity relationship studies identified additional compounds containing a core 2-[(E)-2-phenylvinyl]-8-quinolinol structure that disrupted p53 degradation. Upper panel: Activity of compounds in C33a cells expressing TE6 and p53-Luc. Lower panel: Activity of compounds in HeLa cells expressing p53-Luc. (C) 2-[(E)-2-phenylvinyl]-8-quinolinol compounds rescued p53 degradation. The top 11 compounds in (B) were assayed for restoration of p53-Luc activity in C33a cells expressing TE6, in HeLa cells or in C33a cells expressing dsRed. * indicates P < .001. (D) Compounds containing the core structure restored p53 activity <i>in vitro</i>. Recombinant E6 was incubated with the indicated compounds as well as with cell lysates containing p53-Luc protein. (E) Surface plasmon resonance demonstrated that compound 4 bound to p53 with a K_D of 200 + 52 nM. p53 recombinant proteins were amino terminally linked to a Biacore chip. Compound 4 was run at concentrations from 1–25 μM over the Biacore chip and association and disassociation times recorded. (F) Compound 4 docks with the DNA binding domain pocket of p53. Molecular modeling was performed with MolDock.</p

A High-Throughput Cell-Based Screen Identified a 2-[(E)-2-Phenylvinyl]-8-Quinolinol Core Structure That Activates p53

<div><p>p53 function is frequently inhibited in cancer either through mutations or by increased degradation via MDM2 and/or E6AP E3-ubiquitin ligases. Most agents that restore p53 expression act by binding MDM2 or E6AP to prevent p53 degradation. However, fewer compounds directly bind to and activate p53. Here, we identified compounds that shared a core structure that bound p53, caused nuclear localization of p53 and caused cell death. To identify these compounds, we developed a novel cell-based screen to redirect p53 degradation to the Skip-Cullin-F-box (SCF) ubiquitin ligase complex in cells expressing high levels of p53. In a multiplexed assay, we coupled p53 targeted degradation with Rb1 targeted degradation in order to identify compounds that prevented p53 degradation while not inhibiting degradation through the SCF complex or other proteolytic machinery. High-throughput screening identified several leads that shared a common 2-[(E)-2-phenylvinyl]-8-quinolinol core structure that stabilized p53. Surface plasmon resonance analysis indicated that these compounds bound p53 with a K_D of 200 ± 52 nM. Furthermore, these compounds increased p53 nuclear localization and transcription of the p53 target genes PUMA, BAX, p21 and FAS in cancer cells. Although p53-null cells had a 2.5±0.5-fold greater viability compared to p53 wild type cells after treatment with core compounds, loss of p53 did not completely rescue cell viability suggesting that compounds may target both p53-dependent and p53-independent pathways to inhibit cell proliferation. Thus, we present a novel, cell-based high-throughput screen to identify a 2-[(E)-2-phenylvinyl]-8-quinolinol core structure that bound to p53 and increased p53 activity in cancer cells. These compounds may serve as anti-neoplastic agents in part by targeting p53 as well as other potential pathways.</p></div

The common 2-[(E)-2-phenylvinyl]-8-quinolinol core structure did not cause DNA damage or inhibit proteasome activity.

<p>(A) Compounds did not cause double stranded DNA damage as measured by γH2AX phosphorylation. C33a cells were treated with 30 μM of compounds or cisplatin for 4 hrs, fixed and stained for phosphorylated γH2AX. (B) Quantitation of phosphorylated γH2AX positive nuclei. Results are from 10 fields per 2 replicates of the indicated condition. Impact of compounds on proteasome activity as measured by chymotrypsin-like activity (C) or trypsin-like activity (D). For (C) and (D), C33a cells were incubated for 2 hrs with the indicated compounds at 10 μM or 30 μM and assessed for chymotrypsin and trypsin activity using Proteasome-Glo assay. Paired T test were used to determine significance.</p

High-throughput screen for compounds that stabilized the p53 reporter assay.

<p>(A) TE6 abrogated p53-Luc luciferase activity. C33a cells were infected with Ad-p53-Luc alone or with Ad-Red, Ad-TE6 or Ad-TE7. (B) TE7 abrogated Rb-Ren luciferase activity. C33a cells were infected with Ad-Rb-Ren alone or with Ad-Red, Ad-TE6 or Ad-TE7. (C) TE6 promoted proteasome degradation of the p53-Luc reporter. C33a cells infected with Ad-p53-Luc and the cognate Ad-TE6 or non-cognate Ad-TE7 and treated with different concentrations of the proteasome inhibitor bortezomib or the p53 inhibitor RITA. (D) High throughput screening using the TE6-p53-Luc reporter detected compounds that restored p53 activity. A plot of 465 compounds selected from 158,000 compounds that induced p53-Luc or Rb-Ren activity at least 2-fold above background. p53-Luc activity was normalized to the corresponding Rb-Ren activity and vice versa to account for potential proteasome inhibitors or compounds with cytotoxic activity. The dark grey area indicates compounds that restored p53-Luc activity; the light grey area indicates compounds that restored Rb-Ren activity. Paired t-tests were used to determine significance.</p

Compounds caused the p53 nuclear localization and target gene expression.

<p>(A) Compounds 3 or 4 induced p53 nuclear localization. C33a cells were treated with 30 μM compounds for 8 hrs and p53 localization was assessed by immunofluorescence. Representative images from 2 experiments taken at 40X magnification, scale bar = 20 μM. (B) Quantification of p53 nuclear accumulation in (A). (C) Compounds induced p53 protein accumulation in the nucleus. C33a cells were treated with indicated compounds and 6 hrs later fractionated by differential centrifugation. Cytosolic and nuclear fractions were assessed for p53 protein using Western analysis. Calnexin and Lamin A/C mark the cytosolic and nuclear fractions, respectively. (D) Core compounds induced transcription of the p53 target gene PUMA in cancer cells. C33a or HeLa cells were treated with the indicated compounds and, 8h later, PUMA expression as assessed by qRT-PCR using ΔΔCT for GAPDH normalization. (E, left panel) Compound 4 induced p53 binding to the PUMA promoter in C33a cells. (right panel) Compound 4 induced p53 binding to the PUMA promoter in HeLa cells. Results represent experiments performed in triplicate. Significance was assessed using the Student’s t-test.</p

A multiplexed protein targeted degradation system to identify compounds that target p53.

<p>(A) Scheme for a protein targeted degradation strategy to monitor p53 degradation. The protein targeted degradation strategy was based on the Skp-Cullin-F-box (SCF) ubiquitin ligase complex which is an E3 ubiquitin ligase to which the adapter protein βTrCP binds with the F-box domain (orange square). βTrCP recruits target proteins to the SCF complex using its WD40 domain (red oval). When E6 (orange triangle) replaces the WD40 domain of βTrCP, the TE6 fusion protein recruits p53 for degradation. When E7 (green square) replaces the WD40 domain of βTRCP, the TE7 fusion protein recruits Rb for degradation. p53 and Rb which are fused to firefly (Luc) and renilla (Ren) luciferase reporters, respectively, to monitor protein levels. Inhibitors preventing the p53-E6 or Rb-E7 interaction would cause increased p53 or Rb fusion protein levels and their respective luciferase activities. (B) Scheme for adenoviral vectors Ad-p53 and Ad-TE6. Vectors contained an IRES-EGFP or IRES-dsRED as internal controls for p53 or TE6 expression, respectively. (C) TE6 did not affect EGFP expression that serves as an internal control for the p53-Luc reporter expression. C33a cells were infected with Ad-TE6, Ad-p53 or Ad-Red and analyzed for EGFP and RFP by flow cytometry. (D) TE6 expression caused loss of p53-Luc and endogenous p53 protein levels. C33a cells were infected with combinations of Ad-p53, Ad-TE6, Ad-Red and/or Ad-TE7. Whole cell lysates were assayed for p53 expression, TE6 expression using an anti-FLAG epitope or actin expression.</p

Common core structures were cytotoxic to cancer cells in part through a p53-dependent mechanism.

<p>(A) Compounds sharing the common core structure caused decreased viability in the cancer cell lines HeLa, siHA, and C33a. Cells were treated for 48h with compound 3 and 4 at 30 μM concentrations and viability was assessed with Cell Titer-Glo. (B-C) Core structures are cytotoxic with IC₅₀ less than 10 μM. HeLa (B) and C33a (C) cells were incubated with the indicated compounds or cisplatin for 48 hrs and viability measured by Cell Titer Glo. IC₅₀ was determined. (D) Compound 4 caused decreased viability in MCF7, HCT116, HeLa, C33a and SQ20B cells. (E) siRNA inhibition of p53 expression in C33a cells. Cells were transfected with siRNA targeting p53 or a control scrambled sequence and, 48h later, total RNA was assessed for p53 expression. (F) p53 knockdown abrogated the cytotoxicity of compounds containing the common 2-[(E)-2-phenylvinyl]-8-quinolinol core structure. C33a cells were transfected with siRNAs targeting p53 or a controlled scrambled sequence and, 24h later, were treated with 10 μM of the indicated compounds for 48 hrs. Viability was assessed using Cell Titer Glo. (G) Compound 4 was more toxic to wtHCT116 cells possessing a wild type p53 compared to mutHCT116 possessing a null p53 allele. Data was represented as the percent cell viability of mutHCT116 cells divided by the percent cell viability of wtHCT116 cells. * indicates P value < .01 (H) Compound 4 caused cell death as measured by Annexin V and PI staining. (I) Compound 4 did not affect the cell cycle as assessed by PI staining.</p