Development of cyclin E knockouts in breast cancer by intrabody and RNA interference

Breast cancer poses a serious health risk to women in the United States, warranting further investigation into the mechanisms behind breast cancer tumorigenesis. Previous work has identified deregulated expression of cyclin E, a critical regulator of G₁ to S phase progression in mitosis, to be a key contributor to breast cancer tumorigenesis. Overexpression of cyclin E, and the appearance of low molecular weight isoforms, is seen in nearly all breast cancers, and cyclin E expression appears to become progressively more aberrant with increasing tumor stage and grade. Although activation of critical downstream targets of cyclin E such as Rb could lead to loss of cell cycle regulation, and overexpression of cyclin E can induce genomic instability, there appear to be as of now unidentified cyclin E functions in cell division that could contribute to the aggressive and deadly phenotype associated with cyclin E overexpressing breast cancer.; Toward the identification of biomolecular markers and therapeutic targets for breast cancer, we have applied two molecular strategies, intracellular antibodies (intrabodies) and RNA interference, to establish a model system in which to study the implications of suppressing cyclin E activity on breast cancer tumorigenesis. We hypothesized that de novo synthesis of anti-cyclin E intrabodies, or anti-cyclin E small interfering RNAs (siRNAs), would impede cyclin E function in breast cancer cells, reverting the tumorigenic phenotype and blocking the growth of breast cancer cells.; In our first two studies, we describe the construction of two anti-cyclin E single-chain antibodies (sFvs) and their modification for expression in breast cancer cells as nuclear and cytosol-targeted intrabodies. A recombinant lentivirus vector was used to deliver the anti-cyclin E intrabody gene to SKBR3 cells, but showed no significant affect on the growth of these cells. Our third study describes the identification of anti-cyclin E small interfering RNA sequences within the cyclin E gene that lead to specific and efficient suppression of cyclin E expression using RNA interference. We show that expression of small interfering RNAs targeting cyclin E dramatically inhibits the growth of SKBR3 and MCF-7 breast cancer cell lines, and discuss the future utility of this model system in clarifying the molecular link between cyclin E deregulation and breast cancer tumorigenesis.