The human androgen receptor (AR) is a member of the nuclear receptor superfamily and a major target of drugs used to control prostate cancer. This targeting is based on the AR being a determinant of prostate gland growth and differentiation. For this reason, it is important to understand how the AR is regulated inside cells. Previous studies showed that the AR is very unstable in its unliganded state, pointing to the importance of proteolysis to regulating AR steady state levels. Our studies are focused on the mechanisms underlying AR degradation, and how this process may itself be regulated. In preliminary studies, we show that the AR is degraded by the ubiquitin/proteasome pathway. This process is further shown to be affected by a protein called Chip that associates with molecular chaperones such as Hsp7O and Hsp9O. We also show that transfection of Chip inhibits growth of prostate cancer cells that also express AR. The long term goals of this project are to fully characterize the mechanisms governing AR degradation, and how Chip leads to prostate cancer cell growth inhibition In aim 1, we will determine the signals that target AR for degradation. Our hypothesis is that the signals for this event are based partly on AR conformation and partly on cis-acting sequences. We will first determine the role of protein folding in AR degradation, and then examine the role of a putative cis-acting signal, called a PEST sequence, in AR degradation. We will also test for the role of AR phosphorylation as a signal for its degradation.
In aim 2, we will identify the sites of AR ubiquitinylation and the molecular components that catalyze this reaction. We will determine whether there is a functional relationship between ubiquitinylation, sumoylation, and the PEST sequence. We will also identify the E2 and E3 enzymes that conjugate ubiquitin to AR. Further studies will confirm that these enzymes are expressed in human prostate glandular epithelium.
In aim 3, we will detrermine the mechanism by which Chip leads to arrest of prostate cancer cell growth. We will test whether there is a correlation between Chip effects on cell growth and decreasing AR levels, and we will compare the mechanisms of growth arrest in LNCaP cells and their androgen-independent derivative (LNCaPAI) in terms of apoptosis, cell cycle arrest or necrotic cell death.
