Joseph R. Nevins, PhD
Barbara Levine University Professor of Breast Cancer Genomics

Role of E2F proteins in cell proliferation and cell fate. Our previous work has shown that overexpression of various E2F proteins can override cell growth arrest and induce cells to enter S phase. This is consistent with the fact that E2F accumulation is the ultimate event following growth activation of the G1 cycle dependent kinases, leading to phosphorylation of Rb. Indeed, it would appear that the activation of this pathway following growth stimulation is the key and critical event to allow transition through G1/S of the cell cycle. Indeed, it is now clear that the targets of E2F control include most if not all of the genes encoding DNA replication activities. Various studies now suggest that one E2F family member, the E2F3 product, is particularly important for gene control at G1/S. For instance, immunodepletion of E2F3 activity inhibits the induction of S phase in proliferating cells whereas inhibition of E2F1 activity does not affect S phase.

Although E2F1 may not be particularly important for proliferation, it is required for signaling cell fate through an induction of apoptosis. This E2F1 specific apoptosis coincides with an ability of E2F1 to induce p53 accumulation which can now be understood in the context of the role of E2F1 in inducing p19ARF that controls Mdm2, thus allowing p53 to accumulate. The concept of a specific role for E2F1 in signaling cell fate can also be seen in the cellular response to DNA damage. Our recent work has shown that DNA damage leads to an induction of E2F1 accumulation and we have further shown that this is specific for E2F1, that the induction of E2F1 is dependent on ATM kinase activity, and that the specificity of E2F1 induction reflects a specificity in the phosphorylation of E2F1 by ATM. It thus appears that the cellular response to DNA damage makes use of signals from the Rb/E2F cell cycle pathway, potentially to allow a synergistic activation of p53, or possibly to allow the activation of other p53-independent pathways.

Connecting the action of Ras and Myc with the Rb/E2F pathway. Numerous experiments have demonstrated a critical role for the Myc and Ras oncoproteins in cell proliferation. We have shown that inhibition of Ras activity blocks the activation of G1 CDK activity and prevents activation of the target genes of E2F. We have also shown that Ras alone is not sufficient for S phase but coexpression of Ras with Myc allows the generation of cyclin E-dependent kinase activity and the induction of S phase. In addition, our 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 have now shown that Myc-induced S phase and apoptosis requires distinct E2F activities. The ability of Myc to induce S phase requires E2F2 and E2F3 whereas the ability of Myc to induce apoptosis requires E2F1. Thus, the induction of specific E2F activities is an essential component in the Myc pathway that controls cell proliferation and cell fate decisions. Moreover, the unique requirement of E2F1 in Myc-induced apoptosis points once again to the specific role played by this E2F protein in the control of apoptosis.

Using gene expression profiles to study tumor phenotypes. Prognostic and predictive factors are indispensable tools in the treatment of patients with neoplastic disease. For the most part, such factors rely on a few specific cell surface, histological, or gross pathologic features. Gene expression assays have the potential to supplement what were previously a few distinct features with many thousands of features. We have developed Bayesian regression models that provide predictive capability based on gene expression data derived from DNA microarray analysis of a series of primary breast cancer samples. These patterns have the capacity to discriminate breast tumors on the basis of estrogen receptor (ER) status, and also on the categorized lymph node status. The practical value of such approaches relies on the ability not only to assess relative probabilities of clinical outcomes for future samples, but also to provide an honest assessment of the uncertainties associated with such predictive classifications based on the selection of gene subsets for each validation analysis. This latter point is of critical importance in the ability to apply these methodologies to clinical assessment of tumor phenotype.

Current and Future Work
Specificity of E2F function. A paradigm for specificity of E2F function can be seen from the formation of a specific complex on the adenovirus E2 promoter facilitated by a product of the adenovirus E4 gene. The interaction with E4 is dependent on an E2F domain referred to as the marked box. We have instituted two hybrid screens for cellular proteins that interact with individual E2F family members via this marked box domain. This screen has yielded several transcriptional regulatory proteins that can synergistically activate transcription with a specific E2F. We plan to extend these observations to explore interactions underlying E2F transcriptional specificity, studies that will provide an important opportunity for understanding transcriptional specificity via combinatorial interactions.

The unique response of E2F1 to DNA damage, leading to a stabilization of E2F1, coincides with a specific, ATM-mediated phosphorylation of the E2F1 protein. We will further explore the mechanistic basis for this connection by analyzing the effect of phosphorylation on the interaction of ubiquitin ligase complexes with E2F1. A candidate activity involved in targeting E2F1 is the SCF complex containing protein Skp2.

The identification of specific functions of E2F family members provides an opportunity to explore the mechanistic basis for this specificity. One approach will be the assay of a series of chimeric proteins, in which domains of E2F1 and E2F3 are exchanged. We will measure the ability of the chimeras to induce apoptosis as well as to rescue the proliferation defects of the E2F3 null fibroblasts and ultimately mice by replacement of the wild type gene with the chimeras. Additional assays will measure phosphorylation and stability in response to DNA damage. Finally, we will focus on transcriptional specificity, both through the assay of promoter-specific associations by in vivo chromatin assays as well as the use of transcriptional synergy assays as described below.

Role of Myc in the Rb/E2F pathway. We plan to expand our initial findings demonstrating a role for E2Fs in Myc function, primarily through the use of transgenic mice that express Myc in either the B cell lineage (EmMyc) or the mammary gland (MMTV-myc). These will be crossed into various E2F null backgrounds to explore the in vivo relationship of E2F function with Myc, particularly the role of E2Fs in Myc-mediated tumorigenesis. Ras-dependent control of Myc accumulation. We have demonstrated the importance of multiple Ras-dependent phosphorylation events in the control of Myc protein stability but the precise pathway and sequence of events remains to be determined. Our recent work points to a role for specific dephosphorylation as a key and final step in Myc degradation. We will investigate this further by establishing assays to identify activities triggering this process. We will also explore the possibility that these phosphorylations represent a common mechanism for control of other immediate early proteins including cyclin D and c-Jun, following a growth response.

Gene expression profiling for analysis of oncogenic pathways. Our initial studies of breast cancer, using DNA microarray analysis, revealed patterns of gene expression that could distinguish classes of tumors. Our current and future studies are focused on applying these methodologies for predicting clinically-significant aspects of breast cancer that include metastatic potential as well as response of tumors to standard adjuvant chemotherapy. In parallel, we are employing these methodologies of gene expression analysis to define patterns of gene expression that reflect the activity of specific oncogenic pathways such as the Rb/E2F, Myc, Ras, and others. By so doing, we aim to develop signatures of the deregulated pathway that can then be used to explore the events occurring in human tumor development.