Cell death from ECM-detachment, or anoikis, is a conserved cell death pathway that plays an important role in both development and tumorigenesis. Development of anoikis resistance is a critical requirement for metastatic cancer cells. The human Bit1 protein has been shown to be a powerful regulator of anoikis signaling.

Bit1 is a very enigmatic protein in that it is capable of producing two seemingly opposite phenomena. These are the pro-survival observation of the PTH domain of Bit1 (C-terminus) and the pro-death observation of the cell death domain of Bit1 (N-terminus). Both of these functions that have been seen experimentally and are intrinsically linked to one over-arching structure, the Bit1 protein itself. My task through this thesis has been to understand the essence of the Bit1-cell death pathway and to help determine how Bit1 can elicit a specific but lethal cell death response during anoikis.

It has been previously shown that Bit1 is needed to be in the cytoplasm to be apoptotic. Further, cytoplasmic Bit1 requires binding to the AES protein to execute anoikis signaling. However, it is not known what effect Bit1 binding has on AES and how this binding event can trigger cell death. To address this question, co-transfection experiments were performed with cytoplasmic localized Bit1 (or various truncated Bit1 fragments) and FLAG-AES (Chapter 3). Surprisingly, Bit1 caused a decrease in the amount of AES species in the co-transfected cells, indicating that Bit1 is able to on some level down-regulate the AES protein. This effect was also seen with another Groucho/TLE (Gro/TLE) family member TLE-1. In Chapter 3, I will present data showing that this Bit1-induced down-regulation is actually a degradation pathway. In Chapter 4, I will show evidence that the Gro/TLE proteins are being degraded in a proteasome-dependent manner since proteasome inhibitors where able to rescue both AES and TLE1 protein down-regulation. Taken together, these data show that Bit1 is able to target AES and TLE-1 to the ubiquitin-proteasome system and that during anoikis, these proteins need to be degraded for proper cell death signaling.