The development of an oncogenic state is a complex process involving the accumulation of multiple independent mutations that lead to deregulation of cell signalling pathways central to the control of cell growth and cell fate. The ability to define cancer subtypes, recurrence of disease and response to specific therapies using DNA microarray-based gene expression signatures has been demonstrated in multiple studies. Various studies have also demonstrated the potential for using gene expression profiles for the analysis of oncogenic pathways. Here we show that gene expression signatures can be identified that reflect the activation status of several oncogenic pathways. When evaluated in several large collections of human cancers, these gene expression signatures identify patterns of pathway deregulation in tumours and clinically relevant associations with disease outcomes. Combining signature-based predictions across several pathways identifies coordinated patterns of pathway deregulation that distinguish between specific cancers and tumour subtypes. Clustering tumours based on pathway signatures further defines prognosis in respective patient subsets, demonstrating that patterns of oncogenic pathway deregulation underlie the development of the oncogenic phenotype and reflect the biology and outcome of specific cancers. Predictions of pathway deregulation in cancer cell lines are also shown to predict the sensitivity to therapeutic agents that target components of the pathway. Linking pathway deregulation with sensitivity to therapeutics that target components of the pathway provides an opportunity to make use of these oncogenic pathway signatures to guide the use of targeted therapeutics.

Our recent work has shown that activation of the Ras/Raf/ERK pathway extends the half-life of the Myc protein and thus enhances the accumulation of Myc activity. We have extended these observations by investigating two N-terminal phosphorylation sites in Myc, Thr 58 and Ser 62, which are known to be regulated by mitogen stimulation. We now show that the phosphorylation of these two residues is critical for determining the stability of Myc. Phosphorylation of Ser 62 is required for Ras-induced stabilization of Myc, likely mediated through the action of ERK. Conversely, phosphorylation of Thr 58, likely mediated by GSK-3 but dependent on the prior phosphorylation of Ser 62, is associated with degradation of Myc. Further analysis demonstrates that the Ras-dependent PI-3K pathway is also critical for controlling Myc protein accumulation, likely through the control of GSK-3 activity. These observations thus define a synergistic role for multiple Ras-mediated phosphorylation pathways in the control of Myc protein accumulation during the initial stage of cell proliferation.

The cellular transcription factor E2F, previously identified as a component of early adenovirus transcription, has now been shown to be important in cell proliferation control. E2F appears to be a functional target for the action of the tumor suppressor protein Rb that is encoded by the retinoblastoma susceptibility gene. The disruption of this E2F-Rb interaction, as well as a complex involving E2F in association with the cell cycle-regulated cyclin A-cdk2 kinase complex, may be a common mechanism of action for the oncoproteins encoded by the DNA tumor viruses.

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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 status and also on the categorized lymph node status. Importantly, we assess the utility and validity of such models in predicting the status of tumors in crossvalidation determinations. 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 on the basis of 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.

The stability of c-Myc is regulated by multiple Ras effector pathways. Phosphorylation at Ser 62 stabilizes c-Myc, whereas subsequent phosphorylation at Thr 58 is required for its degradation. Here we show that Ser 62 is dephosphorylated by protein phosphatase 2A (PP2A) before ubiquitination of c-Myc, and that PP2A activity is regulated by the Pin1 prolyl isomerase. Furthermore, the absence of Pin1 or inhibition of PP2A stabilizes c-Myc. A stable c-Myc(T58A) mutant that cannot bind Pin1 or be dephosphorylated by PP2A replaces SV40 small T antigen in human cell transformation and tumorigenesis assays. Therefore, small T antigen, which inactivates PP2A, exerts its oncogenic potential by preventing dephosphorylation of c-Myc, resulting in c-Myc stabilization. Thus, Ras-dependent signalling cascades ensure transient and self-limiting accumulation of c-Myc, disruption of which contributes to human cell oncogenesis.

Over the past decade, studies focusing on the mechanisms controlling cellular proliferation have converged with equally intensive efforts directed at the analysis of oncogenic pathways associated with human cancer. These convergent studies have revealed the central role played by the pathway that controls the activity of the retinoblastoma tumor suppressor protein (Rb), which in turn regulates the E2F transcription factor. In particular, it is now clear that the Rb/E2F pathway is critical in regulating the initiation of DNA replication. It is also clear that the control of the pathway is disrupted in virtually all human cancers. Questions remain, however, as to the specific role played by individual activities within the pathway in the control of cell growth and their participation in the development of cancer.

Several lines of evidence implicate the E2F transcription factor as an important component of cell proliferation control. First, E2F binding sites are found in the promoters of genes responsive to proliferation signals and the level of E2F binding activity increases at a time when many of these genes are activated. Second, the tumour suppressor protein Rb, as well as the related p107 protein, complexes with E2F, resulting in an inhibition of E2F transcriptional activity. Third, oncogenic products of the DNA tumour viruses can dissociate these E2F complexes. We provide here direct evidence that E2F is involved in cellular proliferation control. Specifically, we demonstrate that overexpression of the E2F1 complementary DNA can activate DNA synthesis in cells that would otherwise growth-arrest, with an efficiency that is similar to that achieved by the expression of the adenovirus E1A gene. Moreover, microinjection of the E2F1 cDNA into quiescent cells can induce S-phase entry, whereas two E2F1 mutants, which are unable to transactivate the DHFR and TK promoters, are unable to induce S phase. We conclude that the E2F transcription factor plays an important role in progression into S phase and that this probably coincides with its capacity to stimulate transcription.