Cynthia Timmers

The E2F1–3 transcription factors are essential for cellular proliferation

The retinoblastoma tumour suppressor (Rb) pathway is believed to have a critical role in the control of cellular proliferation by regulating E2F activities. E2F1, E2F2 and E2F3 belong to a subclass of E2F factors thought to act as transcriptional activators important for progression through the G1/S transition. Here we show, by taking a conditional gene targeting approach, that the combined loss of these three E2F factors severely affects E2F target expression and completely abolishes the ability of mouse embryonic fibroblasts to enter S phase, progress through mitosis and proliferate. Loss of E2F function results in an elevation of p21Cip1 protein, leading to a decrease in cyclin-dependent kinase activity and Rb phosphorylation. These findings suggest a function for this subclass of E2F transcriptional activators in a positive feedback loop, through down-modulation of p21Cip1, that leads to the inactivation of Rb-dependent repression and S phase entry. By targeting the entire subclass of E2F transcriptional activators we provide direct genetic evidence for their essential role in cell cycle progression, proliferation and development.

Mouse development with a single E2F activator

The E2F family is conserved from Caenorhabditis elegans to mammals, with some family members having transcription activation functions and others having repressor functions. Whereas C. elegans and Drosophila melanogaster have a single E2F activator protein and repressor protein, mammals have at least three activator and five repressor proteins. Why such genetic complexity evolved in mammals is not known. To begin to evaluate this genetic complexity, we targeted the inactivation of the entire subset of activators, E2f1, E2f2, E2f3a and E2f3b, singly or in combination in mice. We demonstrate that E2f3a is sufficient to support mouse embryonic and postnatal development. Remarkably, expression of E2f3b or E2f1 from the E2f3a locus (E2f3a3bki or E2f3a1ki, respectively) suppressed all the postnatal phenotypes associated with the inactivation of E2f3a. We conclude that there is significant functional redundancy among activators and that the specific requirement for E2f3a during postnatal development is dictated by regulatory sequences governing its selective spatiotemporal expression and not by its intrinsic protein functions. These findings provide a molecular basis for the observed specificity among E2F activators during development.

Inactivation of E2F3 results in centrosome amplification

The E2F family of transcription factors is critical for the control of cell cycle progression. We now show that the specific inactivation of E2F3 in mouse embryo fibroblasts (MEFs) results in a disruption of the centrosome duplication cycle. Loss of E2F3, but not E2F1, E2F2, E2F4, or E2F5 results in unregulated cyclin E-dependent kinase activity, defects in nucleophosmin B association with centrosomes, and premature centriole separation and duplication. Consequently, this defect leads to centrosome amplification, mitotic spindle defects, and aneuploidy. Our findings implicate the E2F3 transcription factor as an important link that orchestrates DNA and centrosome duplication cycles, ensuring the faithful transmission of genetic material to daughter cells.

Enhanced bioavailability of lycopene when consumed as cis-isomers from tangerine compared to red tomato juice, a randomized, cross-over clinical trial

SCOPE Tangerine tomatoes (Solanum lycopersicum) are rich in tetra-cis-lycopene resulting from natural variation in carotenoid isomerase. Our objective was to compare the bioavailability of lycopene from tangerine to red tomato juice, and elucidate physical deposition forms of these isomers in tomatoes by light and electron microscopy. METHODS AND RESULTS Following a randomized cross-over design, subjects (n = 11, 6 M/5 F) consumed two meals delivering 10 mg lycopene from tangerine (94% cis) or red tomato juice (10% cis). Blood was sampled over 12 h and triglyceride-rich lipoprotein fractions of plasma were isolated and analyzed using HPLC-DAD-MS/MS. Lycopene was crystalline in red tomato chromoplasts and globular in tangerine tomatoes. With tangerine tomato juice we observed a marked 8.5-fold increase in lycopene bioavailability compared to red tomato juice (p < 0.001). Fractional absorption was 47.70 ± 8.81% from tangerine and 4.98 ± 1.92% from red tomato juices. Large heterogeneity was observed among subjects. CONCLUSION Lycopene is markedly more bioavailable from tangerine than from red tomato juice, consistent with a predominance of cis-lycopene isomers and presence in chromoplasts in a lipid dissolved globular state. These results justify using tangerine tomatoes as a lycopene source in studies examining the potential health benefits of lycopene-rich foods.

Control of the p53-p21CIP1 Axis by E2f1, E2f2, and E2f3 is essential for G1/S progression and cellular transformation.

The E2F family of transcription factors is believed to have an essential role in the control of cellular proliferation by regulating the transcription of genes involved in cell cycle progression. Previous work has demonstrated that the targeted inactivation of E2f1, E2f2, and E2f3 results in elevated p21CIP1 protein levels, loss of E2F target gene expression, and cell cycle arrest at G1/S and G2/M, suggesting a strict requirement for these E2Fs in the control of normal cellular proliferation. We now demonstrate that E2f1, E2f2, and E2f3 are also required for oncogene-mediated transformation of mouse embryonic fibroblasts. Analysis of synchronized populations of mouse embryonic fibroblasts revealed that the inactivation of p21CIP1 restores the ability of E2f1-3-deficient cells to enter and transit through G1/S (but not G2/M). In contrast, loss of p53 restored the ability of these cells to progress through both G1/S and mitosis, leading to their continued proliferation. The inactivation of p53 (but not p21CIP1) rendered E2f1-3-deficient cells sensitive to transformation and tumorigenesis. These results suggest that the negative regulation of the p53-p21CIP1 axis by the E2F1-3 factors is critical for cell cycle progression and cellular transformation.

The Ras oncogene signals centrosome amplification in mammary epithelial cells through cyclin D1/Cdk4 and Nek2

Centrosome amplification (CA) contributes to carcinogenesis by generating aneuploidy. Elevated frequencies of CA in most benign breast lesions and primary tumors suggest a causative role for CA in breast cancers. Clearly, identifying which and how altered signal transduction pathways contribute to CA is crucial to breast cancer control. Although a causative and cooperative role for c-Myc and Ras in mammary tumorigenesis is well documented, their ability to generate CA during mammary tumor initiation remains unexplored. To answer that question, K-RasG12D and c-Myc were induced in mouse mammary glands. Although CA was observed in mammary tumors initiated by c-Myc or K-RasG12D, it was detected only in premalignant mammary lesions expressing K-RasG12D. CA, both in vivo and in vitro, was associated with increased expression of the centrosome-regulatory proteins, cyclin D1 and Nek2. Abolishing the expression of cyclin D1, Cdk4 or Nek2 in MCF10A human mammary epithelial cells expressing H-RasG12V abrogated Ras-induced CA, whereas silencing cyclin E1 or B2 had no effect. Thus, we conclude that CA precedes mammary tumorigenesis, and interfering with centrosome-regulatory targets suppresses CA.

E2f1-3 Control E2F-target Expression and Cellular Proliferation via a p53-dependent Negative Feedback Loop

ABSTRACT E2F-mediated control of gene expression is believed to have an essential role in the control of cellular proliferation. Using a conditional gene-targeting approach, we show that the targeted disruption of the entire E2F activator subclass composed of E2f1, E2f2, and E2f3 in mouse embryonic fibroblasts leads to the activation of p53 and the induction of p53 target genes, including p21CIP1. Consequently, cyclin-dependent kinase activity and retinoblastoma (Rb) phosphorylation are dramatically inhibited, leading to Rb/E2F-mediated repression of E2F target gene expression and a severe block in cellular proliferation. Inactivation of p53 in E2f1-, E2f2-, and E2f3-deficient cells, either by spontaneous mutation or by conditional gene ablation, prevented the induction of p21CIP1 and many other p53 target genes. As a result, cyclin-dependent kinase activity, Rb phosphorylation, and E2F target gene expression were restored to nearly normal levels, rendering cells responsive to normal growth signals. These findings suggest that a critical function of the E2F1, E2F2, and E2F3 activators is in the control of a p53-dependent axis that indirectly regulates E2F-mediated transcriptional repression and cellular proliferation.

Bone morphogenetic protein 3B silencing in non-small-cell lung cancer

Bone morphogenetic protein 3B (BMP3B) is a member of the TGF-β superfamily. The BMP3B promoter sequence was previously identified as a target for aberrant DNA methylation in non-small-cell lung cancer (NSCLC). Aberrant DNA hypermethylation in the BMP3B promoter is associated with downregulation of BMP3B transcription in both primary human lung cancers as well as lung cancer cell lines. In order to understand the mechanisms of BMP3B silencing in lung cancer, a sample set of 91 primary NSCLCs was used to detect aberrant BMP3B promoter methylation, mutations in the coding sequence of BMP3B, and loss of heterozygosity (LOH). Our results showed that 45 of 91 (or 49.5%) tested primary NSCLCs exhibited increased promoter methylation, and 40% demonstrated LOH in at least one of the flanking microsatellite markers sJRH and D10S196 (63 kb upstream or 3.338 Mbp downstream of BMP3B). The lung cancer cell line A549, a type II alveolar epithelial human lung cancer cell line, is characterized by aberrant DNA promoter methylation. We used retroviral vector constructs containing the BMP3B cDNA to re-express the gene in A549 cells and to investigate the effects on cell growth. No change in the cell growth rate was observed after BMP3B re-expression, as compared to the vector controls. Although the number of colonies formed in anchorage-dependent assays was only slightly decreased, the colony-forming ability of A549 cells after BMP3B expression in anchorage-independent assays in soft agar was significantly reduced to 10% (P<0.005, t-test). Moreover, the in vivo tumorigenicity assay in nude mice indicated that cells re-expressing BMP3B grew significantly slower than cells not expressing BMP3B (P<0.05, t-test). In conclusion, this study provides evidence that BMP3B expression is repressed by different mechanisms in lung cancer, and that the silencing of BMP3B promotes lung tumor development.

Randomized Phase 2 Trial of the Oncolytic Virus Pelareorep (Reolysin) in Upfront Treatment of Metastatic Pancreatic Adenocarcinoma

Pelareorep causes oncolysis in tumor cells with activated Ras. We hypothesized that pelareorep would have efficacy and immunomodulatory activity in metastatic pancreatic adenocarcinoma (MPA) when combined with carboplatin and paclitaxel. A randomized phase 2 study (NCT01280058) was conducted in treatment-naive patients with MPA randomized to two treatment arms: paclitaxel/carboplatin + pelareorep (Arm A, n = 36 evaluable patients) versus paclitaxel/carboplatin (Arm B, n = 37 evaluable patients). There was no difference in progression-free survival (PFS) between the arms (Arm A PFS = 4.9 months, Arm B PFS = 5.2 months, P = 0.6), and Kirsten rat sarcoma viral oncogene (KRAS) status did not impact outcome. Quality-adjusted Time without Symptoms or Toxicity analysis revealed that the majority of PFS time was without toxicity or progression (4.3 months). Patient immunophenotype appeared important, as soluble immune biomarkers were associated with treatment outcome (fractalkine, interleukin (IL)-6, IL-8, regulated on activation, normal T cell expressed and secreted (RANTES), and vascular endothelial growth factor (VEGF)). Increased circulating T and natural killer (NK)-cell subsets were also significantly associated with treatment outcome. Addition of pelareorep was associated with higher levels of 14 proinflammatory plasma cytokines/chemokines and cells with an immunosuppressive phenotype (Tregs, cytotoxic T lymphocyte associated protein 4 (CTLA4)(+) T cells). Overall, pelareorep was safe but does not improve PFS when administered with carboplatin/paclitaxel, regardless of KRAS mutational status. Immunologic studies suggest that chemotherapy backbone improves immune reconstitution and that targeting remaining immunosuppressive mediators may improve oncolytic virotherapy.

Comparison of custom capture for targeted next-generation DNA sequencing.

Targeted, capture-based DNA sequencing is a cost-effective method to focus sequencing on a coding region or other customized region of the genome. There are multiple targeted sequencing methods available, but none has been systematically investigated and compared. We evaluated four commercially available custom-targeted DNA technologies for next-generation sequencing with respect to on-target sequencing, uniformity, and ability to detect single-nucleotide variations (SNVs) and copy number variations. The technologies that used sonication for DNA fragmentation displayed impressive uniformity of capture, whereas the others had shorter preparation times, but sacrificed uniformity. One of those technologies, which uses transposase for DNA fragmentation, has a drawback requiring sample pooling, and the last one, which uses restriction enzymes, has a limitation depending on restriction enzyme digest sites. Although all technologies displayed some level of concordance for calling SNVs, the technologies that require restriction enzymes or transposase missed several SNVs largely because of the lack of coverage. All technologies performed well for copy number variation calling when compared to single-nucleotide polymorphism arrays. These results enable laboratories to compare these methods to make informed decisions for their intended applications.