Effects of activin and cyclin overexpression on Smad 3 function in breast cancer cells

Abstract

22072 Background: Activin, a growth factor in the TGFβ superfamily, triggers the activation of Smad 3, a transcription factor that contributes to G1 cell cycle arrest in breast cancer cells. Overexpression of cyclin E, another key cell cycle regulatory protein, and its low molecular weight (LMW) forms, have been associated with poor prognosis in breast cancer. Cyclin E has been shown to mediate phosphorylation of the Smad 3 linker region by cyclin dependent kinases (cdk), resulting in inhibition of Smad 3 activity. We hypothesize that overexpression of cyclin E may exert tumorigenic effects through the functional inhibition of Smad 3 in breast cancer cells. Methods: Western blotting was used to evaluate the basal protein expression of Smad 3 and phosphorylated Smad 3 (phospho-Smad 3), in vector control and a LMW cyclin E overexpressing MCF-7 breast cancer cell line. To determine the impact of cyclin E overexpression on Smad 3 function, wild-type (WT) or linker region mutated Smad 3 constructs were co-transfected with a Smad 3 reporter into the vector control and the LMW cyclin E overexpressing MCF-7 cells. The MCF-7 cells were also treated with activin or not. Smad 3 function was then evaluated by luciferase reporter assay. Results: MCF-7 cells overexpressing the LMW cyclin E expressed a higher level of phospho-Smad 3 as compared to the vector control cells, while total Smad 3 levels remained unchanged. Expression of the linker region mutated Smad 3 in control cells resulted in greater than three-fold induction in Smad 3 reporter activity compared to the WT Smad 3. This induction was further pronounced in MCF-7 cells transfected with the linker region mutated Smad 3 and treated with activin. Interestingly, expression of the linker region mutated Smad 3 in MCF- 7 cells overexpressing the LMW cyclin E resulted in an eight-fold induction of the Smad 3 reporter, as compared to the WT Smad 3. Conclusions: This study indicates that overexpression of a LMW form of cyclin E may circumvent the growth inhibitory effects of activin/Smad 3 signal transduction through the blockade of Smad 3 function. The overexpression of phospho-Smad 3, found in the LMW cyclin E cells, may be a compensatory mechanism by activin/Smad 3 signaling to surmount the tumorigenic effects of LMW cyclin E in these cells. No significant financial relationships to disclose.