Gustavo Leone

Emerging roles of E2Fs in cancer: an exit from cell cycle control

Mutations of the retinoblastoma tumour suppressor gene (RB1) or components regulating the RB pathway have been identified in almost every human malignancy. The E2F transcription factors function in cell cycle control and are intimately regulated by RB. Studies of model organisms have revealed conserved functions for E2Fs during development, suggesting that the cancer-related proliferative roles of E2F family members represent a recent evolutionary adaptation. However, given that some human tumours have concurrent RB1 inactivation and E2F amplification and overexpression, we propose that there are alternative tumour-promoting activities for the E2F family, which are independent of cell cycle regulation.

Role for E2F in Control of Both DNA Replication and Mitotic Functions as Revealed from DNA Microarray Analysis

ABSTRACT We have used high-density DNA microarrays to provide an analysis of gene regulation during the mammalian cell cycle and the role of E2F in this process. Cell cycle analysis was facilitated by a combined examination of gene control in serum-stimulated fibroblasts and cells synchronized at G1/S by hydroxyurea block that were then released to proceed through the cell cycle. The latter approach (G1/S synchronization) is critical for rigorously maintaining cell synchrony for unambiguous analysis of gene regulation in later stages of the cell cycle. Analysis of these samples identified seven distinct clusters of genes that exhibit unique patterns of expression. Genes tend to cluster within these groups based on common function and the time during the cell cycle that the activity is required. Placed in this context, the analysis of genes induced by E2F proteins identified genes or expressed sequence tags not previously described as regulated by E2F proteins; surprisingly, many of these encode proteins known to function during mitosis. A comparison of the E2F-induced genes with the patterns of cell growth-regulated gene expression revealed that virtually all of the E2F-induced genes are found in only two of the cell cycle clusters; one group was regulated at G1/S, and the second group, which included the mitotic activities, was regulated at G2. The activation of the G2 genes suggests a broader role for E2F in the control of both DNA replication and mitotic activities.

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.

Pten in stromal fibroblasts suppresses mammary epithelial tumours

The tumour stroma is believed to contribute to some of the most malignant characteristics of epithelial tumours. However, signalling between stromal and tumour cells is complex and remains poorly understood. Here we show that the genetic inactivation of Pten in stromal fibroblasts of mouse mammary glands accelerated the initiation, progression and malignant transformation of mammary epithelial tumours. This was associated with the massive remodelling of the extracellular matrix (ECM), innate immune cell infiltration and increased angiogenesis. Loss of Pten in stromal fibroblasts led to increased expression, phosphorylation (T72) and recruitment of Ets2 to target promoters known to be involved in these processes. Remarkably, Ets2 inactivation in Pten stroma-deleted tumours ameliorated disruption of the tumour microenvironment and was sufficient to decrease tumour growth and progression. Global gene expression profiling of mammary stromal cells identified a Pten-specific signature that was highly represented in the tumour stroma of patients with breast cancer. These findings identify the Pten–Ets2 axis as a critical stroma-specific signalling pathway that suppresses mammary epithelial tumours.

Ras Enhances Myc Protein Stability

Various experiments have demonstrated a collaborative action of Myc and Ras, both in normal cell growth control as well as during oncogenesis. We now show that Ras enhances the accumulation of Myc activity by stabilizing the Myc protein. Whereas Myc has a very short half-life when produced in the absence of mitogenic signals, due to degradation by the 26S proteasome, the half-life of Myc increases markedly in growth-stimulated cells. This stabilization is dependent on the Ras/Raf/MAPK pathway and is not augmented by proteasome inhibition, suggesting that Ras inhibits the proteasome-dependent degradation of Myc. We propose that one aspect of Myc-Ras collaboration is an ability of Ras to enhance the accumulation of transcriptionally active Myc protein.

Direct Evidence for Epithelial-Mesenchymal Transitions in Breast Cancer

We developed stromal- and epithelial-specific cre-transgenic mice to directly visualize epithelial-mesenchymal transition (EMT) during cancer progression in vivo. Using three different oncogene-driven mouse mammary tumor models and cell-fate mapping strategies, we show in vivo evidence for the existence of EMT in breast cancer and show that myc can specifically elicit this process. Hierarchical cluster analysis of genome-wide loss of heterozygosity reveals that the incidence of EMT in invasive human breast carcinomas is rare, but when it occurs it is associated with the amplification of MYC. These data provide the first direct evidence for EMT in breast cancer and suggest that its development is favored by myc-initiated events.

Myc requires distinct E2F activities to induce S phase and apoptosis.

Previous work has shown that the Myc transcription factor induces transcription of the E2F1, E2F2, and E2F3 genes. Using primary mouse embryo fibroblasts deleted for individual E2F genes, we now show that Myc-induced S phase and apoptosis requires distinct E2F activities. The ability of Myc to induce S phase is impaired in the absence of either E2F2 or E2F3 but not E2F1 or E2F4. In contrast, the ability of Myc to induce apoptosis is markedly reduced in cells deleted for E2F1 but not E2F2 or E2F3. From this data, we propose that the induction of specific E2F activities is an essential component in the Myc pathways that control cell proliferation and cell fate decisions.

The accumulation of an E2F-p130 transcriptional repressor distinguishes a G0 cell state from a G1 cell state.

Previous studies have demonstrated cell cycle-dependent specificities in the interactions of E2F proteins with Rb family members. We now show that the formation of an E2F-p130 complex is unique to cells in a quiescent, G0 state. The E2F-p130 complex does not reform when cells reenter a proliferative state and cycle through G1. The presence of an E2F-p130 complex in quiescent cells coincides with the E2F-mediated repression of transcription of the E2F1 gene, and we show that the E2F sites in the E2F1 promoter are important as cells enter quiescence but play no apparent role in cycling cells. In addition, the decay of the E2F-p130 complex as cells reenter the cell cycle requires the action of G1 cyclin-dependent kinase activity. We conclude that the accumulation of the E2F-p130 complex in quiescent cells provides a negative control of certain key target genes and defines a functional distinction between these G0 cells and cells that exist transiently in G1.

E2f1–3 switch from activators in progenitor cells to repressors in differentiating cells

In the established model of mammalian cell cycle control, the retinoblastoma protein (Rb) functions to restrict cells from entering S phase by binding and sequestering E2f activators (E2f1, E2f2 and E2f3), which are invariably portrayed as the ultimate effectors of a transcriptional program that commit cells to enter and progress through S phase. Using a panel of tissue-specific cre-transgenic mice and conditional E2f alleles we examined the effects of E2f1, E2f2 and E2f3 triple deficiency in murine embryonic stem cells, embryos and small intestines. We show that in normal dividing progenitor cells E2f1–3 function as transcriptional activators, but contrary to the current view, are dispensable for cell division and instead are necessary for cell survival. In differentiating cells E2f1–3 function in a complex with Rb as repressors to silence E2f targets and facilitate exit from the cell cycle. The inactivation of Rb in differentiating cells resulted in a switch of E2f1–3 from repressors to activators, leading to the superactivation of E2f responsive targets and ectopic cell divisions. Loss of E2f1–3 completely suppressed these phenotypes caused by Rb deficiency. This work contextualizes the activator versus repressor functions of E2f1–3 in vivo, revealing distinct roles in dividing versus differentiating cells and in normal versus cancer-like cell cycles.

Identification of a Novel E2F3 Product Suggests a Mechanism for Determining Specificity of Repression by Rb Proteins

ABSTRACT The tumor suppressor function of Rb is intimately related to its ability to interact with E2F and repress the transcription of E2F target genes. Here we describe a novel E2F product that specifically interacts with Rb in quiescent cells. This novel E2F, which we term E2F3b, is encoded by a unique mRNA transcribed from an intronic promoter within the E2F3 locus. The E2F3b RNA differs from the previously characterized E2F3 RNA, which we now term E2F3a, by the utilization of a unique coding exon. In contrast to the E2F3a product that is tightly regulated by cell growth, the E2F3b product is expressed equivalently in quiescent and proliferating cells. But, unlike the E2F4 and E2F5 proteins, which are also expressed in quiescent cells and form complexes with the p130 protein, the E2F3b protein associates with Rb and represents the predominant E2F-Rb complex in quiescent cells. Thus, the previously described specificity of Rb function as a transcriptional repressor in quiescent cells coincides with the association of Rb with this novel E2F product.