The early G2/M checkpoint and ubiquitination: Implications for breast tumorigenesis

The ubiquitin/proteasome pathway is highly conserved in evolution and is essential for many diverse cellular functions. Many of the short-lived cell cycle regulatory proteins are targets of this proteasome pathway. Unregulated cell cycle proteins are important contributors to tumorigenesis. Efforts to identify and develop drugs that target the ubiquitin/proteasome pathway to treat malignancies have become an intense area of investigation. However, targeted drug design requires a better understanding of the many genes involved in the ubiquitin/proteasome pathway. The first aim of my thesis was to examine a ubiquitin ligase, CHFR (checkpoint with f&barbelow;orkhead-associated and r&barbelow;ing finger) that also functions as part of the early G2/M checkpoint, in breast cancer. This checkpoint delays chromosome condensation in response to microtubule poisons. We found that half of the 24 breast cancer cell lines studied had low CHFR expression. Following nocodazole treatment, breast cancer cells with low CHFR expression failed to arrest in prophase, whereas cells with higher CHFR expression arrested in prophase as expected. Further analysis revealed that some of the nocodazole treated cells may have progressed through mitosis due to lack of any functional mitotic checkpoints (i.e., early G2/M and spindle checkpoints). The second aim of my thesis was to identify novel oncogene candidates on the long arm of chromosome 17 through analysis of regions of amplifications. We detected two distinct amplicons on 17q23 that harbor putative oncogenes and several uncharacterized ESTs specifically expressed in breast tissue. Ubiquitin specific protease 6 (USP6), a gene that mapped more proximal to the 17q23 amplicons, was also significantly overexpressed in breast cancer cell lines. We demonstrated that overexpression of USP6 in NIH3T3 and 293 cells induced an increased growth rate but such overexpression alone could not induce neoplastic transformation in these cells. Overexpression of USP6 and other candidate oncogenes within identified amplicons may be functioning cooperatively during breast carcinogenesis. Additional studies to reveal the expression and function of genes in the ubiquitin/proteasome pathway will help us elucidate the complex interactions of cell-cycle regulation. A better understanding of how this pathway operates should then lead to the development of efficient therapeutical applications.