Recent studies have indicated the existence of tumorigenesis barriers that slow or inhibit the progression of preneoplastic lesions to neoplasia. One such barrier involves DNA replication stress, which leads to activation of the DNA damage checkpoint and thereby to apoptosis or cell cycle arrest, whereas a second barrier is mediated by oncogene-induced senescence. The relationship between these two barriers, if any, has not been elucidated. Here we show that oncogene-induced senescence is associated with signs of DNA replication stress, including prematurely terminated DNA replication forks and DNA double-strand breaks. Inhibiting the DNA double-strand break response kinase ataxia telangiectasia mutated (ATM) suppressed the induction of senescence and in a mouse model led to increased tumour size and invasiveness. Analysis of human precancerous lesions further indicated that DNA damage and senescence markers cosegregate closely. Thus, senescence in human preneoplastic lesions is a manifestation of oncogene-induced DNA replication stress and, together with apoptosis, provides a barrier to malignant progression.

Lineage survival oncogenes are activated by somatic DNA alterations in cancers arising from the cell lineages in which these genes play a role in normal development.1,2 Here we show that a peak of genomic amplification on chromosome 3q26.33, found in squamous cell carcinomas (SCCs) of the lung and esophagus, contains the transcription factor gene SOX2—which is mutated in hereditary human esophageal malformations3 and necessary for normal esophageal squamous development4, promotes differentiation and proliferation of basal tracheal cells5 and co-operates in induction of pluripotent stem cells.6,7,8 SOX2 expression is required for proliferation and anchorage-independent growth of lung and esophageal cell lines, as shown by RNA interference experiments. Furthermore, ectopic expression of SOX2 cooperated with FOXE1 or FGFR2 to transform immortalized tracheobronchial epithelial cells. SOX2-driven tumors show expression of markers of both squamous differentiation and pluripotency. These observations identify SOX2 as a novel lineage survival oncogene in lung and esophageal SCC.

Epidermal growth factor receptor (EGFR) overexpression is observed in a number of malignancies, especially those of esophageal squamous cell origin. However, little is known about the biological functions of EGFR in primary esophageal squamous epithelial cells. Using newly established primary human esophageal squamous epithelial cells as a platform, we overexpressed EGFR through retroviral transduction and established novel threedimensional organotypic cultures. Additionally, EGFR was targeted in a cell type-and tissue-specific fashion to the esophageal epithelium in transgenic mice. EGFR overexpression in primary esophageal keratinocytes resulted in the biochemical activation of Akt and STAT pathways and induced enhanced cell migration and cell aggregation. When established in organotypic culture, EGFR-overexpressing cells had evidence of epithelial cell hyperproliferation and hyperplasia. These effects were also observed in EGFR-overexpressing transgenic mice and the esophageal cell lines established thereof. In particular, EGFR-induced effects upon aggregation appear to be mediated through the relocalization of p120 from the cytoplasm to the membrane and increased interaction with E-cadherin. EGFR modulates cell migration through the up-regulation of matrix metalloproteinase 1. Taken together, the functional effects of EGFR overexpression help to explain its role in the initiating steps of esophageal squamous carcinogenesis. Epidermal growth factor receptor (EGFR)1 is a transmembrane protein receptor with tyrosine kinase activity that triggers numerous signaling pathways (1-3). Activation of the EGFR tyrosine kinase results in the generation of a number of intracellular signals, which culminate in not only cell proliferation but also other processes that are crucial to cancer progression, including angiogenesis, metastatic spread, and the inhibition of apoptosis. These events are mediated through various downstream targets of EGFR (e.g. the serine/threonine kinase Raf and mitogen-activated protein/extracellular signalregulated kinase 1/2). In addition, Ras activation by EGFR is required for a vast array of cellular functions, foremost of which is the regulation of cellular proliferation. Activation of EGFR also results in the activation of the lipid kinase phosphatidylinositol 3-kinase, generating the second messenger phosphatidylinositol 3,4,5-trisphosphate, which in turn activates Akt. We have previously demonstrated that there is differential activation of the Akt isoforms by EGFR in esophageal cancer cells (4). Apart from the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways, EGFR also activates other pathways such as phospholipase-C and its downstream protein kinase cascades, small GTPases such as Rho, and multiple signal transducer and activator of transcription (STAT) isoforms.EGFR activation is not only important in normal cellular processes, but it is frequently altered or overexpressed in many malignancies, especially those of squamous cell origin. Mechanisms that mediate E...

Summary SCFFbx4 was recently identified as the E3 ligase for cyclin D1. We now describe cell cycle-dependent phosphorylation and dimerization of Fbx4 that is regulated by GSK3β and defective in human cancer. We present data demonstrating that a pathway involving Ras-Akt-GSK3β controls the temporal phosphorylation and dimerization of the SCFFbx4 E3 ligase. Inhibition of Fbx4 activity results in accumulation of nuclear cyclin D1 and oncogenic transformation. The importance of this regulatory pathway for normal cell growth is emphasized by the prevalence of mutations in Fbx4 in human cancer that impair dimerization. Collectively, this data reveals that inactivation of the cyclin D1 E3 ligase will likely contribute to cyclin D1 overexpression in a significant fraction of human cancer.

Summary Cyclin D1 elicits transcriptional effects through inactivation of the retinoblastoma protein and direct association with transcriptional regulators. The current work reveals a molecular relationship between cyclin D1/CDK4 kinase and protein arginine methyltransferase 5 (PRMT5), an enzyme associated with histone methylation and transcriptional repression. Primary tumors of a mouse lymphoma model exhibit increased PRMT5 methyltransferase activity and histone arginine methylation. Analyses demonstrate that MEP50, a PRMT5 co-regulatory factor, is a CDK4 substrate, and phosphorylation increases PRMT5/MEP50 activity. Increased PRMT5 activity mediates key events associated with cyclin D1-dependent neoplastic growth including CUL4 repression, CDT1 overexpression, and DNA re-replication. Importantly, human cancers harboring mutations in Fbx4, the cyclin D1 E3 ligase, exhibit nuclear cyclin D1 accumulation and increased PRMT5 activity.