Thymidylate synthase (TS) is an E2F1-regulated enzyme that is essential for DNA synthesis and repair. TS protein and mRNA levels are elevated in many human cancers, and high TS levels have been correlated with poor prognosis in patients with colorectal, breast, cervical, bladder, kidney, and non-small cell lung cancers. In this study, we show that ectopic expression of catalytically active TS is sufficient to induce a transformed phenotype in mammalian cells as manifested by foci formation, anchorage independent growth, and tumor formation in nude mice. In contrast, comparable levels of two TS mutants carrying single point mutations within the catalytic domain had no transforming activity. In addition, we show that overexpression of TS results in apoptotic cell death following serum removal. These data demonstrate that TS exhibits oncogene-like activity and suggest a link between TS-regulated DNA synthesis and the induction of a neoplastic phenotype.
Thymidylate synthase (TS) is an essential enzyme for DNA synthesis and repair and elevated levels of TS have been identified as an important prognostic biomarker for colorectal cancer and several other common human malignancies. In addition, TS gene expression has been linked with cell-cycle regulation and cell proliferation through the ability of retinoblastoma protein to repress the transcriptional activation of E2F target genes such as TS. Therefore, overproduction of TS could participate in the progression to a neoplastic phenotype. Consistent with this model, a recent study has suggested that ectopic TS expression can induce a transformed phenotype in mammalian cells. To investigate the role of deregulated TS activity in tumor development, we generated transgenic mice that express high levels of catalytically active human TS (hTS) exclusively in the pancreas and low levels of hTS in multiple other tissues. Analyses of pancreatic tissue in TS transgenic mice revealed abnormalities within the endocrine pancreas, ranging from pancreatic islet hyperplasia to the detection of islet cell tumors. Overexpression of hTS in murine islets provides a new model to study genetic alterations associated with the progression from normal cells to hyperplasia to islet cell tumors, and suggests that this mouse model may be useful for regulating TS activity in vivo for development of cancer prevention and new therapies.
The overexpression or mutation of tyrosine kinases (TKs), such as the epidermal growth factor receptor (EGFR), can lead to the development of cancer. The most common mutation of the EGFR in glioblastomas is the deletion of exons 2-7 known as the EGFRvIII. This mutant receptor cannot bind EGF but, instead, is constitutively active. The Cbl family of ubiquitin ligases (Cbl, Cbl-b, and Cbl-c) targets the activated EGFR for degradation. As the EGFRvIII is transforming, we investigated whether it could be downregulated by the Cbl proteins. The over-expression of all three Cbl proteins resulted in the ubiquitination and degradation of the EGFRvIII. As with the wild-type EGFR, the TK-binding domain and the RING finger of Cbl-b are sufficient for the downregulation of the EGFRvIII. Also, we found that Cbl-b is recruited to the EGFRvIII and inhibits the transformation of NIH 3T3 cells by the EGFRvIII. Mutation of the Cbl-binding site (Y1045F) in the EGFRvIII inhibits its ubiquitination and downregulation by Cbl-b and enhances its ability to transform. Furthermore, the EGFR TK inhibitor, AG 1478, prevents the downregulation of the EGFRvIII by the Cbl proteins and antagonizes the ability of an immunotoxin directed against the EGFRvIII to kill cells expressing this receptor. In conclusion, the EGFR-vIII does not transform by escaping regulation by Cbl proteins and this activation-induced downregulation of the EGFRvIII has an important role in mediating the toxicity of anti-EGFRvIII immunotoxins.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.